Metastasis inhibitor composition comprising a phospholipid-linked glycosaminoglycan and method for inhibiting metastasis employing the same

ABSTRACT

This invention relates to compounds prepared by linking glycosaminoglycan to phospholipid or lipid, which are expected to exert a pharmacological effect for inhibiting metastasis because of their excellent function to inhibit adhesion of cancer cells to blood vessel endothelial cells and extracellular matrix. This phospholipid- or lipid-linked glycosaminoglycan can be produced for example by: cleaving and oxidizing reducing terminal group of glycosaminoglycan, and allowing an aldehyde group or a lactone compound of the thus-formed derivative or a carboxyl group in the glycosaminoglycan chain to react with a primary amino group of a phospholipid; or linking a glycosaminoglycan derivative to a phospholipid or a lipid by allowing a primary amino group of the derivative to react with a carboxyl group of the phospholipid or lipid. This phospholipid- or lipid-linked glycosaminoglycan is useful as a metastasis inhibitor because it has no toxicity.

This is a Divisional of application Ser. No. 07/847,065 filed Mar. 24,1992 (now U.S. Pat. No. 5,464,942), which is the U.S. National Stageentry under 35 U.S.C. 371 of PCT/JP91/00995, filed Jul. 24, 1991, andnow abandoned.

FIELD OF THE INVENTION

This invention relates to phospholipid- or lipid-linkedglycosaminoglycans, a process for producing the same and metastasisinhibitors.

BACKGROUND OF THE INVENTION

During the process of metastasis development, it is known that a cancercell which has strayed into a blood vessel or lymphoduct adheres to anendothelial cell or to its extracellular matrix (so-called basementmembrane) and the thus adhered cancer cell permeates into theextracellular matrix to develop a new metastatic lesion in the tissue.For example, S. Korach et al. (Cancer Research, 46, 3624-3629, 1986)reported that they have divided cancer cells into a high metastaticgroup and a low metastatic group and through their cancer cell cloningstudies, conducted in vitro adhesion tests of cancer cells to culturedendothelial cells. As a result, the adhesiveness of cancer cells toblood vessel endothelial cells or to their extracellular matrices isclosely related to metastasis of cancer cells because the highmetastatic group cancer cells showed a high adhesiveness while the lowmetastatic cells showed a low adhesiveness.

On the other hand, the peptide sequence GRGDS (Gly-Arg-Gly-Asp-Ser) ofthe cell adhesion moiety of fibronectin, which is a component of theextracellular matrix, competitively inhibits binding between cancercells and the extracellular matrix. Yamada et al. (Science, 233,467-470, 1986) reported that the peptide GRGDS inhibited lung metastasisof B16F10 cells in mice. These results indicate that a substance whichhas cell adhesion inhibitory activity in a small amount could be used asa metastasis inhibitor.

The present invention has been accomplished based on a finding thatcertain types of phospholipid- or lipid-linked glycosaminoglycans caninhibit adhesion of cancer cells to blood vessel endothelial cells andtheir extracellular matrices and, as a result, can inhibit metastasis ofcancer cells.

DESCRIPTION OF THE INVENTION

The present invention relates to phospholipid- or lipid-linkedglycosaminoglycans, a process for producing the same and metastasisinhibitors containing the same or the salts thereof.

As shown in Table 1, glycosaminoglycan is a long chain polysaccharidewhich consists of recurring units of disaccharides or tetrasaccharidesincluding D-glucosamine or D-galactosamine, D-glucuronic acid,L-iduronic acid and/or D-galactose. Examples of known glycosaminoglycansinclude hyaluronic acid, chondroitin, chondroitin sulfate A, chondroitinsulfate C, chondroitin sulfate D, chondroitin sulfate E, chondroitinsulfate K, chondroitin polysulfate, dermatan sulfate, heparin, heparansulfate, keratan sulfate and keratan polysulfate.

                  TABLE 1                                                         ______________________________________                                        Glycosaminoglycan                                                                           Hexosamine   Uronic acid                                        ______________________________________                                        Hyaluronic acid                                                                             GlcNAc       GlcUA                                              (MW, 1,000-10,000,000)                                                        Chondroitin   GalNAc       GlcUA                                              (MW, 1,000-100,000)                                                           Chondroitin sulfate A                                                                       GalNAc (4S)  GlcUA                                              (MW, 1,000-100,000)                                                           Chondroitin sulfate C                                                                       GalNAc (6S)  GlcUA                                              (MW, 1,000-100,000)                                                           Chondroitin sulfate D                                                                       GalNAc (6S)  GlcUA (2S)                                         (MW, 1,000-100,000)                                                           Chondroitin sulfate E                                                                       GalNAc (4S,6S)                                                                             GlcUA                                              (MW, 1,000-100,000)                                                           Chondroitin sulfate K                                                                       GalNAc (4S)  GlcUA (3S)                                         (MW, 1,000-100,000)                                                           Chondroitin polysulfate                                                                     GalNAc (S)   GlcUA (S)                                          (MW, 1,000-150,000)                                                           Dermatan sulfate                                                                            GalNAc (4S)  IduUA, GlcUA                                       (MW, 1,000-20,000)                                                            Heparin       GlcNS (6S)   GlcUA, IduUA (2S)                                  (MW, 1,000-20,000)                                                            Heparan sulfate                                                                             GlcNS (NAc,S)                                                                              GlcUA, IduUA (2S)                                  (MW, 1,000-20,000)                                                            Keratan sulfate                                                                             GlcNAc (6S)  Gal                                                (MW, 1,000-20,000)                                                            Keratan polysulfate                                                                         GlcNAc (6S)  Gal (6S)                                           (MW, 1,000-20,000)                                                            ______________________________________                                         GlcNAc: Nacetyl-D-glucosamine                                                 GalNAc: Nacetyl-D-galactosamine                                               GlcNS: Dglucosamine Nsulfate                                                  GlcUA: Dglucuronic acid                                                       IduUA: Liduronic acid                                                         Gal: Dgalactose                                                               S: Osulfate                                                              

The phospholipid- or lipid-linked glycosaminoglycan of the presentinvention can be used as a salt, preferably with an alkali metal such assodium, potassium or the like, an alkaline earth metal such as calcium,magnesium or the like and an amine such as trialkylamine, pyridine orthe like.

The following are examples of the phospholipid- or lipid-linkedglycosaminoglycans of the present invention.

A phospholipid-linked glycosaminoglycan represented by the followingformula or a salt thereof: ##STR1## wherein P¹ is a phospholipid havinga primary amino group and; (1) GAG is located at the 4-position, R³ islocated at the 3-position, R² is a COOH group and R³ is an OH group whenGAG is a glycosaminoglycan residue of hyaluronic acid, chondroitin,chondroitin sulfate A, C or E, dermatan sulfate or heparin excluding areducing terminal glucuronic acid moiety or when GAG is aglycosaminoglycan residue of dermatan sulfate excluding a reducingterminal iduronic acid moiety,

(2) GAG is located at the 4-position, R³ is located at the 3-position,R² is a COOH group and R³ is an OSO₃ H group when GAG is aglycosaminoglycan residue of chondroitin sulfate K or chondroitinpolysulfate excluding a reducing terminal glucuronic acid moiety,

(3) GAG is located at the 3-position, R³ is located at the 4-position,R² is a CH₂ OH group and R³ is an OH group when GAG is aglycosaminoglycan residue of keratan sulfate excluding a reducingterminal galactose moiety, and

(4) GAG is located at the 3-position, R³ is located at the 4-position,R² is a CH₂ OSO₃ H group and R³ is an OH group when GAG is aglycosaminoglycan residue of keratan polysulfate excluding a reducingterminal galactose moiety.

A phospholipid-linked glycosaminoglycan represented by the followingformula or a salt thereof: ##STR2## wherein P¹ is a phospholipid havinga primary amino group and; (1) R¹ is a NHCOCH₃ group and R³ is an OHgroup when GAG is a glycosaminoglycan residue of hyaluronic acid orchondroitin excluding a reducing terminal hexosamine moiety,

(2) R¹ is a NHCOCH₃ group and R³ is an OSO₃ H group when GAG is aglycosaminoglycan residue of chondroitin sulfate A or K, chondroitinpolysulfate or dermatan sulfate excluding a reducing terminal hexosaminemoiety, and

(3) each of R¹ and R³ is an OH group when GAG is a glycosaminoglycanresidue of keratan sulfate or keratan polysulfate excluding a reducingterminal galactose moiety.

A phospholipid-linked glycosaminoglycan represented by the followingformula or a salt thereof: ##STR3## wherein P¹ is a phospholipid havinga primary amino group and GAG is a glycosaminoglycan residue of keratansulfate or keratan polysulfate excluding a reducing terminal galactosemoiety.

A phospholipid-linked glycosaminoglycan represented by the followingformula or a salt thereof: ##STR4## wherein P¹ is a phospholipid havinga primary amino group and; (1) GAG is located at the 4-position, R³ islocated at the 3-position, R¹ is an OH group, R² is a COOH group and R³is an OH group when GAG is a glycosaminoglycan residue of hyaluronicacid, chondroitin, chondroitin sulfate A, C or E, dermatan sulfate,heparin or heparan sulfate excluding a reducing terminal glucuronic acidmoiety or when GAG is a glycosaminoglycan residue of dermatan sulfateexcluding a reducing terminal iduronic acid moiety,

(2) GAG is located at the 4-position, R³ is located at the 3-position,R¹ is an OSO₃ H group, R² is a COOH group and R³ is an OH group when GAGis a glycosaminoglycan residue of chondroitin sulfate D excluding areducing terminal glucuronic acid moiety or when GAG is aglycosaminoglycan residue of heparin or heparan sulfate excluding areducing terminal iduronic acid moiety,

(3) GAG is located at the 4-position, R³ is located at the 3-position,R¹ is an OH group, R² is a COOH group and R³ is an OSO₃ H group when GAGis a glycosaminoglycan residue of chondroitin sulfate K excluding areducing terminal glucuronic acid moiety,

(4) GAG is located at the 4-position, R³ is located at the 3-position,at least one of R¹ and R³ is an OSO₃ H group, while the other is an OHgroup, and R² is a COOH group when GAG is a glycosaminoglycan residue ofchondroitin polysulfate excluding a reducing terminal glucuronic acidmoiety,

(5) GAG is located at the 3-position, R³ is located at the 4-position,each of R¹ and R³ is an OH group and R² is a CH₂ OH group when GAG is aglycosaminoglycan residue of keratan sulfate excluding a reducingterminal galactose moiety,

(6) GAG is located at the 3-position, R³ is located at the 4-position,each of R¹ and R³ is an OH group and R² is a CH₂ OSO₃ H group when GAGis a glycosaminoglycan residue of keratan polysulfate excluding areducing terminal galactose moiety,

(7) GAG is located at the 3-position, R³ is located at the 4-position,R¹ is an NHCOCH₃ group, R² is a CH₂ OH group and R³ is an OH group whenGAG is a glycosaminoglycan residue of hyaluronic acid or chondroitinexcluding a reducing terminal hexosamine moiety,

(8) GAG is located at the 3-position, R³ is located at the 4-position,R¹ is an NHCOCH₃ group, R² is a CH₂ OH group and R³ is an OSO₃ H groupwhen GAG is a glycosaminoglycan residue of chondroitin sulfate A or K ordermatan sulfate excluding a reducing terminal hexosamine moiety,

(9) GAG is located at the 3-position, R³ is located at the 4-position,R¹ is an NHCOCH₃ group, R² is a CH₂ OSO₃ H group and R³ is an OH groupwhen GAG is a glycosaminoglycan residue of chondroitin sulfate C or Dexcluding a reducing terminal hexosamine moiety,

(10) GAG is located at the 3-position, R³ is located at the 4-position,R¹ is an NHCOCH₃ group, R² is a CH₂ OSO₃ H group and R³ is an OSO₃ Hgroup when GAG is a glycosaminoglycan residue of chondroitin sulfate Eexcluding a reducing terminal hexosamine moiety,

(11) GAG is located at the 3-position, R³ is located at the 4-position,R¹ is an NHCOCH₃ group, R² is a CH₂ OH group and R³ is an OSO₃ H group,or R² is a CH₂ OSO₃ H group and R³ is an ON group or an OSO₃ H group,when GAG is a glycosaminoglycan residue of chondroitin polysulfateexcluding a reducing terminal hexosamine moiety,

(12) GAG is located at the 4-position, R³ is located at the 3-position,R¹ is an NHSO₃ H group, R² is a CH₂ OSO₃ H group and R³ is an OH groupwhen GAG is a glycosaminoglycan residue of heparin excluding a reducingterminal hexosamine moiety,

(13) GAG is located at the 4-position, R³ is located at the 3-position,R¹ is an NHCOCH₃ group or an NHSO₃ H group, R² is a CH₂ OH group when R³is an OSO₃ H group, or R² is a CH₂ OSO₃ H group when R³ is an OH groupor an OSO₃ H group, when GAG is a glycosaminoglycan residue of heparansulfate excluding a reducing terminal hexosamine moiety,

(14) GAG is located at the 4-position, R³ is located at the 3-position,R¹ is an NHCOCH₃ group, R² is a CH₂ OSO₃ H group and R³ is an OH groupwhen GAG is a glycosaminoglycan residue of keratan sulfate or keratanpolysulfate excluding a reducing terminal hexosamine moiety.

A phospholipid- or lipid-linked glycosaminoglycan represented by thefollowing formula or a salt thereof: ##STR5## wherein P² is aphospholipid or a lipid, m is an integer of 1 to 8 and l is an integerof 1 to 10, and;

(1) GAG is located at the 4-position, R³ is located at the 3-position,R² is a COOH group and R³ is an OH group when GAG is a glycosaminoglycanresidue of hyaluronic acid, chondroitin, chondroitin sulfate A, C or E,dermatan sulfate, heparin or heparan sulfate excluding a reducingterminal glucuronic acid moiety or when GAG is a glycosaminoglycanresidue of dermatan sulfate excluding a reducing terminal iduronic acidmoiety,

(2) GAG is located at the 4-position, R³ is located at the 3-position,R² is a COOH group and R³ is an OSO₃ H group when GAG is aglycosaminoglycan residue of chondroitin sulfate K or chondroitinpolysulfate excluding a reducing terminal glucuronic acid moiety,

(3) GAG is located at the 3-position, R³ is located at the 4-position,R² is a CH₂ OH group and R³ is an OH group when GAG is aglycosaminoglycan residue of keratan sulfate excluding a reducingterminal galactose moiety, and

(4) GAG is located at the 3-position, R³ is located at the 4-position,R² is a CH₂ OSO₃ H group and R³ is an OH group when GAG is aglycosaminoglycan residue of keratan polysulfate excluding a reducingterminal galactose moiety.

A phospholipid- or lipid-linked glycosaminoglycan represented by thefollowing formula or a salt thereof: ##STR6## wherein GAG, R¹ and R³ areas defined in the foregoing formula (II), and m, l and P² are as definedin the foregoing formula (V).

A phospholipid- or lipid-linked glycosaminoglycan represented by thefollowing formula or a salt thereof: ##STR7## wherein GAG, R¹, R² and R³are as defined in the foregoing formula (IV), and m, l and P² are asdefined in the foregoing formula (V).

A phospholipid-linked glycosaminoglycan represented by the followingformula or a salt thereof: ##STR8## wherein P¹ is a phospholipid havinga primary amino group and n is an integer not more than the number ofcarboxyl groups contained in glycosaminoglycan, and;

(1) each of R¹ and R³ is an OH group when GAG is a glycosaminoglycanchain of hyaluronic acid, chondroitin, chondroitin sulfate A, C or E, ordermatan sulfate,

(2) R¹ is an OSO₃ H group and R³ is an OH group when GAG is aglycosaminoglycan chain of chondroitin sulfate D,

(2) R¹ is an OH group and R³ is an OSO₃ H group when GAG is aglycosaminoglycan chain of chondroitin sulfate K,

(4) at least one of R¹ and R³ is an OSO₃ H group while the other one isan OH group when GAG is a glycosaminoglycan chain of chondroitinpolysulfate, and

(5) R¹ is an OH group or an OSO₃ H group and R₃ is an OH group when GAGis a glycosaminoglycan chain of heparin or heparan sulfate.

Preferred molecular weights of the glycosaminoglycans are listed inTable 1.

The phospholipid having a primary amino group represented by P¹ in theforegoing formulae (I), (II), (III), (IV) and (VIII) is a compoundrepresented by the formula: ##STR9## wherein each of R⁴ and R⁵ ishydrogen, --CH═CHR⁶ or --COR⁷ (each of R⁶ and R⁷ is a C₆₋₂₄ alkyl group)and Y is --CH₂ CH₂ NH-- or ##STR10## Particularly preferred arecompounds in which either of R⁴ and R⁵ is a --COR⁷ group such ashexadecanoyl or octadecanoyl or in which R⁴ is a --CH═CHR⁶ group and R⁵is a --COR⁷ group.

The phospholipid or lipid represented by P² in the foregoing formulae(V), (VI) and (VII) is a compound represented by the formula: ##STR11##wherein R⁸ is hydrogen, R⁹ is an alkyl group, R¹⁰ is --CH═CHR⁶ or--COR⁷, wherein R⁶ and R⁷ are the same as above and X is --CH₂ CH₂ N⁺(CH₃)₃ or an inositol residue. Particularly preferred are a lipidrepresented by the formula (X) or (XI) in which either of R⁸ and R⁹ is a--COR⁷ group such as hexadecanoyl or octadecanoyl or in which R⁸ ishydrogen and R⁹ is a --COR⁷ group, or a phospholipid represented by theformula (XII) or (XIII) in which R¹⁰ is a --COR⁷ group.

According to the present invention, the aforementioned phospholipid- orlipid-linked glycosaminoglycans are produced by the processes as listedbelow.

A process for producing a phospholipid-linked glycosaminoglycanrepresented by the foregoing formula (I) or a salt thereof whichcomprises the steps of; reducing and cleaving the reducing terminalgroup of a glycosaminoglycan represented by the formula (I-1) ##STR12##thereby obtaining a reduced product represented by the formula (I-2)##STR13## oxidizing the reduced product to obtain an oxidized product ofthe formula (I-3) ##STR14## allowing an aldehyde group in the oxidizedproduct to react with a primary amino group of a phospholipid, whereinGAG, R² and R³ in the formulae (I-1), (I-2) and (I-3), are as defined inthe formula (I).

A process for producing a phospholipid-linked glycosaminoglycanrepresented by the formula (II) or a salt thereof which comprises thesteps of;

reducing and cleaving the reducing terminal group of a glycosaminoglycanrepresented by the formula (II-1) ##STR15## thereby obtaining a reducedproduct represented by the formula (II-2) ##STR16## oxidizing thereduced product to obtain an oxidized product of the formula (II-3)##STR17## allowing an aldehyde group in the oxidized product to reactwith a primary amino group of a phospholipid, wherein GAG, R¹ and R³ inthe formulae (II-1), (II-2) and (II-3), are as defined in the foregoingformula (II) and R is an OH or OSO₃ H group.

A process for producing a phospholipid-linked glycosaminoglycanrepresented by the formula (III) or a salt thereof which comprises thesteps of; oxidizing the reduced product represented by the formula(II-2) to obtain an oxidized product of the formula (11) ##STR18##wherein GAG is as defined in the formula (III), and allowing an aldehydegroup in the oxidized product to react with a primary amino group of aphospholipid.

A process for producing a phospholipid-linked glycosaminoglycanrepresented by the formula (IV) or a salt thereof which comprises thesteps of:

oxidizing and cleaving the reducing terminal group of aglycosaminoglycan represented by the formula (12) ##STR19## therebyobtaining an oxidized product represented by the formula (13) ##STR20##lactonizing the oxidized product to obtain a lactone represented by theformula (14) ##STR21## allowing the thus-obtained lactone to react witha primary amino group of a phospholipid, wherein GAG, R¹, R² and R³ inthe formulae (12), (13) and (14), are as defined in the formula (IV) andA is an alkali metal,

A process for producing a phospholipid- or lipid-linkedglycosaminoglycan represented by the formula (V) or a salt thereof whichcomprises the steps of;

allowing an aldehyde compound represented by the formula (I-3) to reactwith an alkylene diamine to obtain a derivative represented by theformula (15) ##STR22## wherein GAG, R², R³ and m are as defined in theformula (V), separately allowing a phospholipid or a lipid to react witha dicarboxylic acid or a dicarboxylic acid anhydride to obtain aphospholipid or lipid derivative having a carboxyl group, and

allowing a primary amino group in the derivative of the formula (15) toreact with the carboxyl group in the phospholipid or lipid derivative.

A process for producing a phospholipid- or lipid-linkedglycosaminoglycan represented by the formula (VI) or a salt thereofwhich comprises the steps of;

allowing an aldehyde compound represented by the formula (II-3) to reactwith an alkylene diamine to obtain a derivative represented by theformula (16) ##STR23## wherein GAG, R¹, R³ and m are as defined in theformula (VI), separately allowing a phospholipid or a lipid to reactwith a dicarboxylic acid or a dicarboxylic acid anhydride to obtain aphospholipid or lipid derivative having a carboxyl group, and

allowing a primary amino group in the derivative of the formula (16) toreact with the carboxyl group in the phospholipid or lipid derivative.

A process for producing a phospholipid- or lipid-linkedglycosaminoglycan represented by the formula (VII) or a salt thereofwhich comprises the steps of;

allowing a lactone compound represented by the formula (14) to reactwith an alkylene diamine to obtain a derivative represented by theformula (17) ##STR24## wherein GAG, R¹, R², R³ and m are as defined inthe formula (VII),

separately allowing a phospholipid or a lipid to react with adicarboxylic acid or a dicarboxylic acid anhydride to obtain aphospholipid or lipid derivative having a carboxyl group, and

allowing a primary amino group in the derivative of the formula (17) toreact with the carboxyl group in the phospholipid or lipid derivative.

A process for producing a phospholipid-linked glycosaminoglycanrepresented by the formula (VIII) or a salt thereof which comprisesallowing a primary amino group of a phospholipid to react with, in thepresence of a condensing agent, a carboxyl group of a glycosaminoglycanrepresented by the formula (18) ##STR25## wherein GAG, R¹, R³ and n areas defined in formula (VIII).

A process for producing a phospholipid-linked glycosaminoglycanrepresented by the formula (VIII) or a salt thereof which comprises thesteps of;

activating a carboxyl group of a glycosaminoglycan represented by theformula (18), and

allowing the activated carboxyl group to react with a primary aminogroup of a phospholipid.

The processes for producing phospholipid- or lipid-linkedglycosaminoglycans of the present invention are described in detailbelow.

Limited oxidation of reducing terminal group

In this process, the reducing terminal uronic acid, galactose orhexosamine moiety of a glycosaminoglycan is partially oxidized andcleaved to form an aldehyde group and the thus-formed aldehyde group issubjected to reductive alkylation reaction with a primary amino group ofa phospholipid to give a phospholipid-linked glycosaminoglycan. Thereaction scheme of this process is described below.

(A) In the case where glucuronic or iduronic acid in the reducingterminal sugar is subjected to the reaction: ##STR26## wherein R³ is asdefined above and P¹ is a phospholipid having a primary amino group.

In the case of using, as the starting material, hyaluronic acid,chondroitin, chondroitin sulfate A, chondroitin sulfate C, chondroitinsulfate E, chondroitin sulfate K, chondroitin polysulfate, dermatansulfate and heparin, represented by the formula (1) having the reducingterminal D-glucuronic acid or L-iduronic acid in which an OH group islinked to the 2-position carbon, atom, a phospholipid-linkedglycosaminoglycan represented by the formula (I-a) is produced inaccordance with the above reaction scheme.

(B) In the case where glucosamine or galactosamine in the reducingterminal sugar is subjected to the reaction: ##STR27## wherein R³ is asdefined above and P¹ is a phospholipid having a primary amino group.

In the case of using, as the starting material, hyaluronic acid,chondroitin, chondroitin sulfate A, chondroitin sulfate K, chondroitinpolysulfate and dermatan sulfate, represented by the formula (4) havingglucosamine or galactosamine as the reducing terminal group in which aCH₂ OH group is linked to the 5-position carbon atom, aphospholipid-linked glycosaminoglycan represented by the formula (II-a)is produced in accordance with the above reaction scheme.

(C) In the case where galactose in the reducing terminal sugar issubjected to the reaction: ##STR28## wherein R³ is as defined above andP¹ is a phospholipid having a primary amino group.

In the case of using keratan sulfate and keratan polysulfate representedby the above formula (7); as the starting material having galactose asthe reducing terminal sugar, a phospholipid-linked glycosaminoglycanrepresented by the formula (I-b), (II-b) or (III) is produced inaccordance with the above reaction scheme.

In the above processes (A), (B) and (C), the reducing terminal sugarmoieties of glycosaminoglycans represented by formulae (1), (4) and (7)are first subjected to reduction cleavage to obtain correspondingcompounds (2), (5) and (8).

Usable as a reducing agent in the reduction step is an alkali salt ofboron hydride such as sodium boron hydride, sodium cyanoboron hydride orthe like.

As a solvent for use in the above reduction reaction, water or a 0.05%borate buffer (pH 8.3) may be used.

The reduction reaction may be effected at a temperature of from 10° to30° C., preferably from 15° to 25° C.

The amount of the reducing agent, though varying depending on its type,ranges from 5 to 50 equivalents, preferably from 25 to 30 equivalents,per mole of compound (1), (4) or (7).

The thus obtained compounds of formulae (2), (5) and (8) are thensubjected to partial oxidation to form aldehyde compounds represented byformulae (3), (6), (9), (10) and (11).

Usable as an oxidation agent in the oxidation reaction is an alkali saltof periodic acid such as sodium periodate, potassium periodate or thelike.

The amount of the oxidation agent ranges from 1 to 10 equivalents,preferably from 3 to 6 equivalents, per mole of compound (2), (5) or(8). The oxidation reaction may be effected at a temperature of from 0°to 10° C., preferably from 0° to 4° C.

Each of the thus-formed aldehyde compounds (3), (6), (9), (10) and (11)can be reacted with a primary amino group of a phospholipid inaccordance with the known reductive alkylation. Thus, thephospholipid-linked glycosaminoglycans of the present inventionrepresented by the formulae (I-a), (II-a), (I-b), (II-b) and (III) areobtained.

Examples of phospholipids to be used in the above reaction includeL-(α-phosphatidyl)ethanolamine, DL-phosphatidyl-L-serine, ethanolamineplasmalogen, serine plasmalogen and the like.

The reductive alkylation reaction for the production of the compoundsrepresented by the formulae (I-a), (II-a), (I-b), (II-b) and (III) maybe effected by mixing the aldehyde compound (3), (6), (9), (10) or (11)and a phospholipid dissolved in chloroform or the like uniformly in asolvent such as water, 0.05M phosphate buffer (pH 7.0) ordimethylformamide and allowing the mixture to react at a temperature offrom 15° to 60° C., and simultaneously or thereafter carrying out areduction reaction using a reducing agent such as sodium cyanoboronhydride or the like.

Examples of compounds which are produced by the limited reducingterminal group oxidation are shown in Table A.

                  TABLE A                                                         ______________________________________                                        Com-                                                                          pound                                                                         No.   Formula          Glycosaminoglycan material                             ______________________________________                                        I-(1)                                                                                ##STR29##       hyaluronic acid, chondroitin, chondroitin sulfate                             A, C or E, dermatan sulfate, heparin, heaparan                                sulfate                                                I-(2)                                                                                ##STR30##       chondroitin sulfate K, chondroitin polysulfate         I-(3)                                                                                ##STR31##       keratan sulfate                                        I-(4)                                                                                ##STR32##       keratan polysulfate                                    II-(1)                                                                               ##STR33##       hyaluronic acid, chondroitin                           II-(2)                                                                               ##STR34##       chondroitin sulfate A or K, chondroitin                                       polysulfate, dermatan sulfate                          II-(3)                                                                               ##STR35##       keratan sulfate, keratan polysulfate                   III                                                                                  ##STR36##       keratan sulfate, keratan polysulfate                   ______________________________________                                    

Lactonization of reducing terminal group

In this process, the reducing terminal uronic acid, galactose orhexosamine moiety of a glycosaminoglycan is subjected to oxidation tocleave the reducing terminal sugar moiety and the cleaved product islactonized and reacted with a primary amino group of a phospholipid toobtain a phospholipid-linked glycosaminoglycan. This reaction scheme isillustrated below. ##STR37## wherein each of R¹, R² and R³ is as definedabove, P¹ is a phospholipid having a primary amino group and A is analkali metal.

According to this process, a glycosaminoglycan represented by formula(12) is firstly subjected to oxidation to cleave its reducing terminalmoiety, thereby obtaining a carboxyl compound represented by formula(13).

Usable as a starting material are compounds represented by the aboveformula (12) including hyaluronic acid, chondroitin, chondroitin sulfateA, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E,chondroitin sulfate K, chondroitin polysulfate, dermatan sulfate,heparin, heparan sulfate, keratan sulfate and keratan polysulfate.

Iodine, bromine or the like may be used in the oxidizing step as theoxidation agent.

The amount of the oxidation agent ranges from 2 to 20 equivalents,preferably from 5 to 15 equivalents, per mole of the compound of formula(12).

Water or a 0.05M phosphate buffer (pH 7.0) may be used as the solvent inthe oxidation reaction.

The oxidation reaction may be effected at a temperature of from 0° to40° C., preferably from 15° to 20° C.

The thus obtained compound of the formula (13) is then subjected to acidtreatment to form a lactone compound represented by formula (14).

A strongly acidic cation exchange resin such as Dowex 50, Amberlite IR120 or the like may be used in the acid treatment.

The thus-formed lactone compound of formula (14) is then allowed toreact with a phospholipid to produce a phospholipid-linkedglycosaminoglycan of the present invention represented by formula (IV).

The same phospholipid compounds as described in the foregoing limitedreducing terminal group oxidation process may be used in this reactionstep.

The reaction of the lactone compound of formula (14) with a phospholipidfor the production of the compound represented by formulae (IV) may beeffected by dissolving the lactone compound of formula (14) in a solventsuch as water, 0.05M phosphate buffer (pH 7.0) or dimethylformamide, andmixing the solution with a phospholipid dissolved in chloroform or thelike uniformly, and allowing the mixture to react at a temperature offrom 5° to 80° C., preferably from 30° to 60° C.

Examples of compounds which are produced by the reducing terminal grouplactonization process are shown in Table B.

                                      TABLE B                                     __________________________________________________________________________    Compound                                                                      No.   Formula           Glycosaminoglycan material                            __________________________________________________________________________    IV-(1)                                                                               ##STR38##        hyaluronic acid, chondroitin, chondroitin sulfate                             A, C or E, dermatan sulfate, heparin, heaparan                                sulfate                                               IV-(2)                                                                               ##STR39##        chondroitin sulfate D, heparin, heparin sulfate       IV-(3)                                                                               ##STR40##        chondroitin sulfate K                                 IV-(4)-a                                                                             ##STR41##        chondroitin polysulfate                               IV-(4)-b                                                                             ##STR42##        chondroitin polysulfate                               IV-(4)-c                                                                             ##STR43##        chondroitin polysulfate                               IV-(5)                                                                               ##STR44##        keratan sulfate                                       IV-(6)                                                                               ##STR45##        keratan polysulfate                                   IV-(7)                                                                               ##STR46##        hyaluronic acid, chondroitin                          IV-(8)                                                                               ##STR47##        chondroitin sulfate A or K, dermantan sulfate         IV-(9)                                                                               ##STR48##        chondroitin sulfate C or D                            IV-(10)                                                                              ##STR49##        chondroitin sulfate E                                 IV-(11)-a                                                                            ##STR50##        chondroitin polysulfate                               IV-(11)-b                                                                            ##STR51##        chondroitin polysulfate                               IV-(11)-c                                                                            ##STR52##        chondroitin polysulfate                               IV-(12)                                                                              ##STR53##        heparin                                               IV-(13)-a                                                                            ##STR54##        heparan sulfate                                       IV-(13)-b                                                                            ##STR55##        heparan sulfate                                       IV-(13)-c                                                                            ##STR56##        heparan sulfate                                       IV-(14)                                                                              ##STR57##        keratan sulfate, keratan polysulfate                  __________________________________________________________________________

Amidation of reducing terminal group

In this process, each of the aldehyde compounds represented by formulae(3), (6), (9) and (10) and the lactone compound represented by formula(14) is allowed to react with an alkylenediamine compound to obtain aglycosaminoglycan derivative having a primary amino group in itsreducing terminal group. The thus-obtained glycosaminoglycan derivativehaving a primary amino group is then allowed to react with aphospholipid or lipid derivative having carboxyl group such that theprimary amino group and the carboxyl group are linked together. Thus, aphospholipid- or lipid-linked glycosaminoglycan is produced. Thereaction scheme of this process is illustrated below. ##STR58## whereineach of R¹, R² and R³ is as defined above and P² is a phospholipid or alipid.

A glycosaminoglycan derivative having a primary amino group in itsreduction terminus, as represented by the above formula (15), (16) or(17), is obtained by allowing each of compounds (3), (6), (9) and (10)prepared by the aforementioned limited reducing terminal oxidationprocess, and compound (14) prepared by the aforementioned reducingterminal lactonization process to react with an alkylenediamine compoundin the presence of a reducing agent.

An alkylenediamine compound usable in this reaction may be selected fromcompounds represented by the formula

    NH.sub.2 --(CH.sub.2).sub.m --NH.sub.2

wherein m is an integer of from 1 to 8.

Sodium cyanoboron hydride or the like may be used as the reducing agent.

The amount of the reducing agent ranges from 10 to 100 moles per mole ofthe glycosaminoglycan to be used in the reaction system.

Water or a 0.05M phosphate buffer may be used as the reaction solvent.

The reaction may be effected at a temperature of from 0° to 60° C.,preferably from 4° to 25° C.

A phospholipid or lipid derivative having a carboxyl group may beobtained by allowing a phospholipid or lipid compound having a hydroxylgroup in its glycerol structure to react with a dicarboxylic acid or adicarboxylic acid anhydride.

Examples of the phospholipid or lipid compound to be used in thisreaction include monoacylglycerol, diacylglycerol,lysophosphatidylcholine, lysophosphatidylinositol, ether lipids, etherphospholipids and the like.

Succinic acid, glutaric acid, adipic acid or the like may be used as thedicarboxylic acid.

Maleic anhydride, succinic anhydride, fumaric anhydride or the like maybe used as the dicarboxylic acid anhydride.

1-ethyl-3-(dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide orthe like may be used as the condensing agent.

Chloroform, acetanilide, dimethylformamide or the like may be used asthe reaction solvent.

The reaction temperature may range from 0° to 60° C. when a dicarboxylicacid is used in the presence of a condensing agent, or from 20° to 80°C. when a dicarboxylic acid anhydride is used.

The reaction of a glycosaminoglycan derivative having a primary aminogroup in its reducing terminal group with a phospholipid or lipidderivative having a carboxyl group may be effected by first activatingthe carboxyl group in the phospholipid or lipid derivative in accordancewith the well known means in the field of peptide chemistry, and thenallowing the resulting activated compound to react with theglycosaminoglycan derivative.

Activation of the carboxyl group in the phospholipid or lipid derivativemay be effected by converting the carboxyl group into an active esterthrough reaction of the phospholipid or lipid derivative withN-hydroxysuccinimide, p-nitrophenol, N-hydroxybenzotriazole,N-hydroxypiperidine, N-hydroxysuccinamide, 2,4,5-tri-chlorophenol or thelike in the presence of a condensing agent.

Chloroform, acetonitrile, dimethylformamide or the like or a mixturethereof may be used as the reaction solvent.1-ethyl-3-(dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide orthe like may be used as the condensing agent.

The reaction may be effected at a temperature of from 0° to 60° C.

The thus-obtained phospholipid or lipid derivative in which the carboxylgroup has been activated is then allowed to react with theglycosaminoglycan derivative of formulae (15), (16) or (17) having aprimary amino group to obtain the phospholipid or lipid-linkedglycosaminoglycans of formulae (V), (VI) and (VII). The solvent used inthis reaction is chloroform, acetonitrile, dimethylformamide or amixture thereof. The reaction temperature ranges from 0° to 60° C.

Illustrative examples of compounds which are produced by the abovereducing terminal group amination process are shown in Table C.

                                      TABLE C                                     __________________________________________________________________________    (RNH(CH.sub.2).sub.mNHCO(CH.sub.2).sub.lCOP.sup.2)                            Compound                                                                      No.   Formula           Glycosaminoglycan material                            __________________________________________________________________________    V-(1)                                                                                ##STR59##        hyaluronic acid, chondroitin, chondroitin sulfate                             A, C or E, dermatan sulfate, heparin, heaparan                                sulfate                                               V-(2)                                                                                ##STR60##        chondroitin sulfate K, chondroitin polysulfate        V-(3)                                                                                ##STR61##        keratan sulfate                                       V-(4)                                                                                ##STR62##        keratan polysulfate                                   VI-(1)                                                                               ##STR63##        hyaluronic acid, chondroitin                          VI-(2)                                                                               ##STR64##        chondroitin sulfate A or K, chondroitin                                       polysulfate, dermantan sulfate                        VI-(3)                                                                               ##STR65##        keratan sulfate keratan polysulfate                   VII-(1)                                                                              ##STR66##        hyaluronic acid, chondroitin, chondroitin sulfate                             A, C or E, dermatan sulfate, heparin, heaparan                                sulfate                                               VII-(2)                                                                              ##STR67##        chondroitin sulfate D, heparin, heparin sulfate       VII-(3)                                                                              ##STR68##        chondroitin sulfate K                                 VII-(4)-a                                                                            ##STR69##        chondroitin polysulfate                               VII-(4)-b                                                                            ##STR70##        chondroitin polysulfate                               VII-(4)-c                                                                            ##STR71##        chondroitin polysulfate                               VII-(5)                                                                              ##STR72##        keratan sulfate                                       VII-(6)                                                                              ##STR73##        keratan polysulfate                                   VII-(7)                                                                              ##STR74##        hyaluronic acid, chondroitin                          VII-(8)                                                                              ##STR75##        chondroitin sulfate A or K, dermantan sulfate         VII-(9)                                                                              ##STR76##        chondroitin sulfate C or D                            VII-(10)                                                                             ##STR77##        chondroitin sulfate E                                 VII-(11)-a                                                                           ##STR78##        chondroitin polysulfate                               VII-(11)-b                                                                           ##STR79##        chondroitin polysulfate                               VII-(11)-c                                                                           ##STR80##        chondroitin polysulfate                               VII-(12)                                                                             ##STR81##        heparin                                               VII-(13)-a                                                                           ##STR82##        heparan sulfate                                       VII-(13)-b                                                                           ##STR83##        heparan sulfate                                       VII-(13)-c                                                                           ##STR84##        heparan sulfate                                       VII-(14)                                                                             ##STR85##        keratan sulfate, keratan polysulfate                  __________________________________________________________________________

Application of condensing agent

Each member of the glycosaminoglycans, excluding keratan sulfate andkeratan polysulfate, contains D-glucuronic acid or L-iduronic acid asthe uronic acid moiety, and each of these acids has a carboxyl grouplinked to its 5-position carbon atom.

In this process, a phospholipid-linked glycosaminoglycan is produced byallowing the uronic acid carboxyl group to react with a primary aminogroup of a phospholipid in the presence of a condensing agent.

The reaction scheme of this process is illustrated below. ##STR86##wherein each of R¹, R³, n and P¹ is as defined above.

Compounds represented by formula (18) to be used as the startingmaterial are selected from hyaluronic acid, chondroitin, chondroitinsulfate A, chondroitin sulfate C, chondroitin sulfate D, chondroitinsulfate E, chondroitin sulfate K, chondroitin polysulfate, dermatansulfate, heparin and heparan sulfate.

Any of the compounds described in the foregoing illustration of thelimited reducing terminal group oxidation process may be used as thephospholipid.

Examples of the condensing agent include diethylcarbodiimide,diisopropylcarbodiimide, methylpropylcarbodiimide,dicyclohexylcarbodiimide, hexamethylenecarbodiimide,heptanemethylenecarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-meso-p-toluenesulfonate,1-t-butyl-3-(3-dimethylaminopropyl)carbodiimide, diphenylcarbodiimide,4,4'-dinitrodiphenylcarbodiimide, di-p-tolylcarbodiimide,bis-(trimethylsilyl)carbodiimide and the like.

The condensing agent may be used in an amount of from 10 to 100 molesper mole of a phospholipid or lipid to be used.

The reaction may be effected at a temperature of from 4° to 60° C.,preferably from 15° to 25° C., in a solvent such as dimethylformamide,chloroform or a mixture thereof.

Illustrative examples of compounds which are produced by thecondensation process are shown in Table D.

                                      TABLE D                                     __________________________________________________________________________    Compound                                                                      No.   Formula           Glycosaminoglycan material                            __________________________________________________________________________    VIII-(1)                                                                             ##STR87##        hyaluronic acid, chondroitin, chondroitin sulfate                             A, C or E, dermatan sulfate                           VIII-(2)                                                                             ##STR88##        chrondroitin sulfate D                                VIII-(3)                                                                             ##STR89##        chondroitin sulfate K                                 VIII-(4)-a                                                                           ##STR90##        chondroitin polysulfate                               VIII-(4)-b                                                                           ##STR91##        chondroitin polysulfate                               VIII-(4)-c                                                                           ##STR92##        chondroitin polysulfate                               VIII-(5)-a                                                                           ##STR93##        heparin, heparan sulfate                              VIII-(5)-b                                                                           ##STR94##        heparin, heparan sulfate                              __________________________________________________________________________

Activation of glycosaminoglycan

In this process, similar to the case of the aforementioned condensingagent-applied process, the phospholipid-linked glycosaminoglycan (VIII)is produced by activating the uronic acid carboxyl group and thenbinding the activated carboxyl group to a primary amino group in aphospholipid.

The same glycosaminoglycan compounds and phospholipid compounds asdescribed in the foregoing condensing agent-applied process may be usedin this process.

Activation of the carboxyl group in the uronic acid moiety of aglycosaminoglycan compound may be effected by well known means in thefield of peptide chemistry, for example, by converting the carboxylgroup into an active ester through reaction of the glycosaminoglycancompound with N-hydroxysuccinimide, p-nitrophenol,N-hydroxybenzotriazole, N-hydroxypiperidine, N-hydroxysuccinamide,2,4,5-trichlorophenol or the like in the presence of a condensing agent.

The carboxyl group of the uronic acid moiety may be subjected to thereaction as a form of amine salt such as of tri(n-butyl)amine salt,triethylamine salt, pyridine salt or the like.

Dimethylformamide, pyridine, dimethylsulfoxide or the like may be usedas the reaction solvent.

1-ethyl-3-(dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide orthe like may be used as the condensing agent.

The reaction may be effected at a temperature of from 0° to 60° C.,preferably from 4° to 20° C.

By allowing the thus obtained carboxyl group-activated glycosaminoglycanto react with a phospholipid, a phospholipid-linked glycosaminoglycan ofthe formula (VIII) is obtained.

This reaction may be effected by allowing the activatedglycosaminoglycan to react with a phospholipid at a temperature of from0° to 90° C., preferably from 25° to 60° C. in a solvent such asdimethylformamide, chloroform or a mixture thereof.

The contents of phospholipid or lipid portions in the phospholipid- orlipid-linked glycosaminoglycans of the present invention represented byformulae (I) to (VIII) may range from 0.005 to 50%, preferably from 2 to10%.

Separation and purification of the phospholipid- or lipid-linkedglycosaminoglycans obtained by the aforementioned processes may becarried out for instance in the following manner. The final reactionsolution in each procedure is mixed with ethanol which has beensaturated with sodium acetate and the resulting precipitate is filteredout to remove unreacted phospholipid or lipid. The thus-separatedprecipitates or subjected to hydrophobic chromatography and the carrieris washed with an aqueous solution of a salt such as ammonium acetate,ammonium chloride, sodium chloride or the like to remove unreactedglycosaminoglycan. Thereafter, the phospholipid- or lipid-linkedglycosaminoglycan absorbed to the carrier is eluted with an aqueoussolution of 10 to 50% methanol.

The metastasis inhibitor of the present invention may be preparedpreferably by mixing each of the phospholipid- or lipid-linkedglycosaminoglycans represented by formulae (I) to (VIII), or itspharmacologically acceptable salt, with solid or liquid carriers ordiluents for medical use, that is, additive agents such as fillers,stabilizers and the like.

Since the salt form of the phospholipid- or lipid-linkedglycosaminoglycan is soluble in water, it is suitable to formulate itinto injectable solutions. The amount of the active ingredient in themedical preparation may be varied within the range of from 1 to 90% byweight based on the weight of the carrier.

The active compound may be orally administered in the form of granules,fine granules, powders, tablets, capsules, pills or solutions, as wellas bulk powders, or administered by intravenous, intramuscular orsubcutaneous injection. The active compound may also be used as externalpreparations such as suppositories, ointments, cataplasmas, plasters,liniments, lotions and the like. Also, it may be made into powder forinjection use which is dissolved in an appropriate liquid upon use.

For the preparation of pharmaceuticals containing the phospholipid- orlipid-linked glycosaminoglycans of the present invention or their salts,any organic or inorganic, and solid or liquid carrier or diluent may beused provided that such additives are acceptable for oral, intestinal,parenteral or topical administration. Carriers to be used in the presentinvention include water, gelatin, lactose, starch, magnesium stearate,talc, animal and plant oils, benzyl alcohol, gum, polyalkylene glycol,petroleum resins, coconut oil, lanolin and other carriers for medicaluse. In addition, levels of stabilizers, moistening agents, emulsifyingagents, as well as salts for adjusting osmotic pressure or maintainingappropriate pH value of the pharmaceutical preparations.

In the case of granules, fine granules, powders, tablets or capsules,the pharmaceutical preparation may contain the active ingredient of thepresent invention preferably in an amount of from 5 to 80% by weight,while 1 to 30% by weight may be preferable in the case of solutions.Further, preferred contents of the active ingredient may be in the rangeof from 1 to 10% by weight in the case of injections, from 1 to 50% byweight in the case of suppositories, and from 0.1 to 10% by weight inthe case of ointments, cataplasmas and the like for use in topicaladministration.

Clinical dose may preferably be in the range of from 100 to 2,000 mg interms of the amount of the active ingredient per day per adult in thecase of oral administration, though the dose may be varied depending onages and symptoms. Preferably, the above daily dose may be administeredonce a day or two or three times a day by dividing the dose accordingly.

When used as injections, preferred dose may be in the range of from 100to 1,000 mg in terms of the amount of the active ingredient per day peradult. When used as ointments, cataplasmas and the like, thesepreparations with the aforementioned contents of the active ingredientmay be applied to an affected part in an appropriate amount.

With regard to acute toxicity of the active ingredient, animal testswere carried out in the following manner. Four-week-old male and femaleSic-ddy mice were preliminarily fed for one week and, when the malesgrew to weigh 23 to 30 g and the females to weigh 20 to 25 g, HA1-PPEADP(lot No. 300) and CS(S3)-PPEADP (lot No. 302-2; both PPEADPs aredescribed later) were dissolved in a physiological saline ordained byThe Pharmacopoea of Japan to a concentration of 5% and intraperitoneallyadministered to the mice. The intraperitoneal administration was appliedto this test because of its most frequent generation of the toxicitysymptoms. 10 males and 10 females were used in each test group. As aresult, it was found that the LD₅₀ value in each test plot was 2,000mg/kg or higher, thus proving the safety of the compound of the presentinvention as a drug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 6 show data on the hydrophobic chromatography ofphospholipid- or lipid-linked glycosaminoglycans produced in Example1-(2)-1), Example 2-(2)-1), Example 2-(3), Example 4-(1), Example 5-(1)and Example 5-(2), respectively.

BEST MODE TO PRACTICE THE INVENTION

The present invention is described in detail below with reference to thefollowing Examples which are not construed to limit the scope of thepresent invention.

In the following examples, contents of phosphorus, phospholipid or lipidand glycosaminoglycan (GAG) in phospholipid- or lipid-linkedgylcosaminoglycan were measured in the following manner.

Measuring Method

1. Determination of GAG

(1) GAG containing uronic acid: Carbazole sulfuric acid method(Bitter-Muir method) (Analytical Biochemistry, 4, 330-334, 1962).

(2) Keratan sulfate or keratan polysulfate containing galactose:Anthrone method (Biochem. J., 50, 298-303, 1952).

2. Determination of phospholipid or lipid

(1) Phosphorus: Molybdenum blue method (Inorganic Applied ColorimetricAnalysis, vol. 4, pp. 130-135 (representative editor, Shiro Hirano;published by Kyoritsu Shuppan).

(2) Fatty acid: A 10 to 50 mg portion of GAG-lipid is dissolved in 10 mlof 1N sodium hydroxide solution and hydrolyzed at 100° C. for 1 hour.The resulting reaction mixture is adjusted to an acidic pH with 1Nhydrochloric acid solution and extracted with chloroform, and thechloroform phase is washed with water. After drying with dehydratedGlauber's salt, the solvent is removed under a reduced pressure. Theresulting residue is sealed in a tube together with an appropriatevolume of methanol containing 3% HCl (gas), heated at 100° C. for 3hours and then extracted three times with petroleum ether. The resultingpetroleum ether phase is washed three times with water to removecontaminated hydrochloric acid. After drying with Glauber's salt, theresulting product is concentrated under a reduced pressure and subjectedto the following gas-liquid chromatography.

Gas-liquid chromatography (GLC)

GC-15A (Shimadzu Corp.)

Loading material: PEG-HT 5% Uniport HP 60/80 (Gasukuro Kogyo Inc.)

Operation condition: gasification chamber, 350° C.

Column temperature: 190° to 200° C.

Column: 3φ×2 m

Flow rate: N₂ 45 ml/min

EXAMPLE 1 Preparation of Phospholipid-linked Glycosaminoglycan byLimited Oxidation of Reducing Terminal Group

(1) Preparation of reducing terminal group-limitedly oxidizedglycosaminoglycan

1) Preparation of reducing terminal residue-cleaved hyaluronic acid

A 2,000 mg portion of hyaluronic acid (HA1; MW, 10,000; cockscomborigin) was dissolved in 200 ml of 0.05M borate buffer (pH 8.3). Afteradding 182 mg of sodium boron hydride, the resulting mixture wasincubated at room temperature for 5 hours to effect the reaction. Thereaction mixture was adjusted to pH 4.5 with acetic acid and then mixedwith ethanol to form a precipitate. The thus-obtained precipitate waswashed with ethanol to give 1,800 mg of reducing terminalresidue-cleaved hyaluronic acid (R-HA1; lot No. 100).

2) Preparation of reducing terminal group-limitedly oxidized hyaluronicacid

A 1,700 mg portion of R-HA1 (lot No. 100) was dissolved in 250 ml of 40mM imidazole (pH 6.5). After adding 139.96 mg of sodium periodate at 0°C., the resulting mixture was incubated at the same temperature for 1hour to effect the reaction. Ethanol was added to the reaction mixtureto form a precipitate. The thus-obtained precipitate was washed withethanol to give 1,600 mg of reducing terminal group-limitedly oxidizedhyaluronic acid (O-HA; lot No. 200).

3) Preparation of other reducing terminal group-limitedly oxidizedglycosaminoglycans (O-GAG)

Reducing terminal residue-cleaved glycosaminoglycans (R-GAG) wereprepared according to the above procedure 1) under the conditions shownin Table E, using each of the following starting materials: hyaluronicacid (HA5; MW, 50,000: HA15; MW, 150,000: cockscomb origin), chondroitin(CH; MW, 15,000; sulfuric acid-removed product from chondroitin sulfateA with acidic methanol solution), chondroitin sulfate C (CS (S1); MW,10,000: CS (S3); MW, 30,000: CS (S6); MW, 60,000: shark cartilageorigin), chondroitin sulfate A (CS (W); MW, 30,000: shark cartilageorigin), dermatan sulfate (DS; MW, 15,000; swine skin origin), heparin(Hep; MW, 15,000; swine small intestine origin), heparan sulfate (ES;MW, 15,000; bovine kidney origin) and keratan sulfate (KS; MW, 15,000;bovine cornea origin). The thus obtained R-GAG samples were subjected tothe above procedure 2) under the conditions shown in Table F to producereducing terminal group-limitedly oxidized glycosaminoglycans (O-GAG).

                  TABLE E                                                         ______________________________________                                                            Reaction condition                                                                          Yield                                       Lot No.  Product    GAG/NaBH.sub.4 (mg/mg)                                                                      (mg)                                        ______________________________________                                          100-2  R-HA5      5000/94.58    4720                                          100-3  R-HA15     1000/6.31     971                                         101      R-CH       1000/63.05    867                                         102      R-CS (S1)  1000/94.58    880                                           102-2  R-CS (S3)  1000/31.50    897                                           102-3  R-CS (S6)  1000/15.76    869                                         103      R-CS (W)   1000/31.50    823                                         104      R-DS       150/9.46      130                                         105      R-Hep      1000/63.05    772                                         106      R-HS        40/2.55       35                                         107      R-KS        20/1.28        14.6                                      ______________________________________                                    

                  TABLE F                                                         ______________________________________                                        Lot                 Reaction condition                                                                           Yield                                      No.      Product    R-GAG/NaIO.sub.4 (mg/mg)                                                                     (mg)                                       ______________________________________                                          200-2  O-HA5      4500/77.0      4310                                         200-3  O-HA15     900/5.14       815                                        201      O-CH       800/45.65      766                                        202      O-CS (S1)  800/68.48      715                                          202-2  O-CS (S3)  800/22.83      774                                          202-3  O-CS (S6)  800/11.41      699                                        203      O-CS (W)   800/22.83      697                                        204      O-DS       100/5.71        82                                        205      O-Hep      700/39.95      666                                        206      O-HS       30/1.71         22                                        207      O-KS       10/0.57         7                                         ______________________________________                                    

(2) Preparation of L-(α-phosphatidyl)ethanolamine dipalmitoyl-linkedglycosaminoglycan (GAG-PPEADP)

1) Preparation of L-(α-phosphatidyl)ethanolamine dipalmitoyl-linkedhyaluronic acid ##STR95##

1,000 mg of lot No. 200 O-HA was dissolved in 100 ml of 0.05M phosphatebuffer (pH 7.0) and 69.2 ml of a chloroform/methanol solvent system(2:1) containing L-(α-phosphatidyl)ethanolamine dipalmitoyl (PPEADP) (1mg/ml) was added thereto. The resulting mixture was further mixed withmethanol to make the mixture into a uniform solution, and the solutionwas incubated at 50° C. for 1 hour. After adding 25 mg of sodiumcyanoboron hydride to the resulting reaction mixture, incubation wascontinued at 50° C. for 2 hours, followed by concentration under areduced pressure. To the concentrate was added five volumes of aceticacid-saturated ethanol to form a precipitate and the precipitate wasrecovered by filtration. The thus-obtained precipitate was dissolved in0.3M ammonium chloride solution, applied to a hydrophobicchromatographic column (TSK gel Phenyl Toyopearl 650M; 400 ml), washedthoroughly with 0.3 M ammonium chloride solution and then eluted with30% methanol aqueous solution. The reaction product of interest wasfound in the 30% methanol fraction, while unreacted HA1 was found in theunabsorbed and washed fractions. The 30% methanol-eluted fraction wasconcentrated under a reduced pressure, desalted by dialysis and thenfreeze-dried to obtain white powder of lot No. 300.

Yield: 40 mg

PPEADP content: 6.21%

Hyaluronic acid content: 62.12%

Hydrophobic chromatogram: Shown in FIG. 1

Hydrophobic chromatography was carried out under the followingconditions.

Column: TSK gel Phenyl 5 PW (7.5φ×7.5 cm)

Solvent: 0-5 min, 0.3M ammonium chloride solution

5-50 min, 30% methanol solution

Flow rate: 0.5 ml/min

Pressure: 7 kg/0.5 cm²

Fraction volume: 1 ml/tube

Detection: OD₂₂₀ nm

Sample: 100 μl (1 mg/ml solution in 0.3M ammonium chloride)

2) Preparation of other phospholipid-linked glycosaminoglycans

Phospholipid-linked glycosaminoglycans were prepared in accordance withthe above procedure (2)-1) from the O-GAG samples shown in Table F andPPEADP under conditions shown in Table G. Results of the analysis of thethus-obtained products are shown in Table G.

                  TABLE G                                                         ______________________________________                                                          Reaction                                                                      condition                                                                     O-GAG/                                                      Lot               PPEADP/     Yield                                                                              PPEADP GAG                                 No.  Product      NaBH.sub.3 CN                                                                             (mg) (%)    (%)                                 ______________________________________                                        300-2                                                                              R-HA5-PPEADP 1000/13.84/5.03                                                                           42   1.33   63.43                               300-3                                                                              R-HA15-PPEADP                                                                              700/3.23/1.17                                                                             35   0.46   63.35                               301  CH-PPEADP     700/32.29/11.73                                                                          30   4.27   59.10                               302  CS(S1)-PPEADP                                                                               700/48.44/17.60                                                                          36   5.89   63.04                               302-2                                                                              CS(S3)-PPEADP                                                                              700/16.15/5.89                                                                            29   2.22   65.52                               302-3                                                                              CS(S6)-PPEADP                                                                              500/5.77/2.09                                                                             20   1.07   67.13                               303  CS(W)-PPEADP 500/11.53/4.19                                                                            22   2.23   67.48                               304  DS-PPEADP    50/2.31/0.84                                                                              3.7  4.21   66.10                               305  Hep-PPEADP   500/23.07/8.38                                                                            3.8  4.30   74.65                               306  HS-PPEADP    20/0.92/0.34                                                                              3.3  4.09   68.40                               307  KS-PPEADP     7/0.33/0.12                                                                              0.5  3.97   66.24                               ______________________________________                                    

EXAMPLE 2 Preparation of Phospholipid-linked Glycosaminoglycan byLactonization of Reducing Terminal Group

(1) Preparation of reducing terminal group-oxidized glycosaminoglycan

1) Preparation of reducing terminal group-oxidized hyaluronic acid

500 mg of hyaluronic acid (HA1; MW, 10,000; cockscomb origin) wasdissolved in 10 ml of water, and the solution was mixed with 5 mlmethanol solution of 0.1M iodine and incubated at room temperature for 6hours to effect the reaction. To the resulting reaction mixture wasadded about 5 ml of 0.1N potassium hydroxide to decolor free iodinemolecules. Potassium acetate-saturated ethanol was added to theresulting solution to form a precipitate and the precipitated productwas collected by filtration, washed thoroughly with ethanol and thendried under a reduced pressure. Thus, 423 mg of reducing terminalgroup-oxidized hyaluronic acid (lot No. 400) was obtained. Reducingsugar was not detected in the product when checked by Somogyi-Nelsonmethod.

2) Preparation of reducing terminal group-lactonized hyaluronic acid

400 mg of the lot No. 400 reducing terminal group-oxidized hyaluronicacid was dissolved in 10 ml of water, and the solution was passedthrough 50 ml of a column of a strongly acidic ion exchange resin (Dowex50(H⁺)) spending 1 hour. Thus, a solution containing 390 mg of reducingterminus-lactonized hyaluronic acid was obtained. Reducing sugar was notdetected in the solution when checked by Somogyi-Nelson method.

The thus-obtained solution was neutralized with tri-n-butylamine andsubsequently freeze-dried to obtain 400 mg of tri-n-butylamine salt ofreducing terminus-lactonized hyaluronic acid (lot No. 500).

3) Preparation of other reducing terminus-lactonized glycosaminoglycans

Reducing terminal group-oxidized glycosaminoglycans were preparedaccording to the above procedure under conditions shown in Table H,using each of the following starting materials: chondroitin (CH; MW,15,000), chondroitin sulfate C (CS (S1); MW, 10,000: CS (S3); MW,30,000: and CS (S6); MW, 60,000): dermatan sulfate (DS; MW, 15,000),heparin (Hep; MW, 15,000) and heparan sulfate (HS; MW, 15,000). Thethus-obtained samples were subjected to the above procedure 2) underconditions shown in Table I to produce reducing terminalgroup-lactonized glycosaminoglycans.

                  TABLE H                                                         ______________________________________                                                         Reaction condition                                           Lot              GAG/0.1M I.sub.2 /                                                                           Yield                                                                              Somogyi-                                 No.   Product    0.1N KOH (mg/ml/ml)                                                                          (%)  Nelson                                   ______________________________________                                        401   CH-COOK    1000/13.4/13.4 828  -                                        402   CS(S1)-COOK                                                                              1000/19.8/19.8 901  -                                        402-2 CS(S3)-COOK                                                                              1000/3.3/3.3   895  -                                        402-3 CS(S6)-COOK                                                                              1000/4.95/4.95 913  -                                        404   DS-COOK     100/0.67/0.67  91  -                                        405   Hep-COOK   1000/6.7/6.7   902  -                                        406   HS-COOK     100/1.34/1.34  88  -                                        ______________________________________                                         *Somogyi-Nelson: presence (+) or absence (-) of reducing sugar determined     by SomogyiNelson method.                                                 

                  TABLE I                                                         ______________________________________                                                         Reaction condition                                           Lot              GAG-COOK/      Yield                                                                              Somogyi-                                 No.  Product     Dowex 50 (H.sup.+) (mg/ml)                                                                   (%)  Nelson                                   ______________________________________                                        501  CH-lactone  800/400        780  -                                        502  CS(S1)-lactone                                                                            900/450        805  -                                        502-2                                                                              CS(S3)-lactone                                                                            800/400        850  -                                        502-3                                                                              CS(S6)-lactone                                                                            900/450        887  -                                        504  DS-lactone   90/100         96  -                                        505  Hep-lactone 900/400        946  -                                        506  HS-lactone  80/40           72  -                                        ______________________________________                                         *Somogyi-Nelson: presence (+) or absence (-) of reducing sugar determined     by SomogyiNelson method.                                                 

(2) Preparation of L-(α-phosphatidyl)ethanolamine dipalmitoyl-linkedglycosaminoglycan (GAG-PPEADP)

1) Preparation of L-(α-phosphatidyl)ethanolamine dipalmitoyl-linkedhyaluronic acid ##STR96##

400 mg of lot No. 500 reducing terminus-lactonized hyaluronic acid wasdissolved in 200 ml of dimethylformamide and 27.6 mg of PPEADP dissolvedin chloroform was added thereto. The resulting mixture was incubated at70° C. for 2 hours. After removing chloroform from the reaction mixtureby distillation, excess volume of sodium acetate aqueous solution wasadded to the residue to make the reaction product into sodium salt.Sodium acetate-saturated ethanol was added thereto to form a precipitateand the thus-formed precipitate was collected by filtration. Theprecipitate was dissolved in 0.3M ammonium acetate solution andsubjected to purification in accordance with the procedure of Example1-(2) to obtain 36 mg of the desired product (lot No. 600).

Phosphorus content: 0.30%

PPEADP content: 6.44%

Hyaluronic acid content: 82.37%

Hydrophobic chromatogram: Shown in FIG. 2.

Measuring conditions are the same as described above.

(2) Preparation of other L-(α-phosphatidyl)ethanolaminedipalmitoyl-linked glycosaminoglycans

These samples were prepared from the reducing terminus-lactonizedglycosaminoglycans shown in Table I and PPEADP in accordance with theabove procedure (2)-1) under conditions shown in Table J. Results of theanalysis of the thus-obtained products are shown in Table K.

                  TABLE J                                                         ______________________________________                                                             Reaction condition                                       Lot No.  Product     GAG-lactone/PPEADP (mg/mg)                               ______________________________________                                        601      CH-PPEADP   700/32.3                                                 602      CS(S1)-PPEADP                                                                             800/55.4                                                 602-2    CS(S3)-PPEADP                                                                             400/9.26                                                 602-3    CS(S6)-PPEADP                                                                             800/9.00                                                 604      DS-PPEADP    90/4.15                                                 605      Hep-PPEADP   800/36.91                                               606      HS-PPEADP    70/3.31                                                 ______________________________________                                    

                  TABLE K                                                         ______________________________________                                                             Yield    PPEADP GAG                                      Lot No.  Product     (mg)     (%)    (%)                                      ______________________________________                                        601      CH-PPEADP   70.2     4.30   90.90                                    602      CS(S1)-PPEADP                                                                             88.0     6.41   85.17                                    602-2    CS(S3)-PPEADP                                                                             20       2.01   89.70                                    602-3    CS(S6)-PPEADP                                                                             56.2     1.08   92.00                                    604      DS-PPEADP   4.5      4.00   90.66                                    605      Hep-PPEADP  24       4.11   90.01                                    606      HS-PPEADP   5.74     4.22   88.21                                    ______________________________________                                    

(3) Production of phosphotidylserin stearoylpalmitoyl-linked chondroitinsulfate C ##STR97##

400 mg of lot No. 502-2 reducing terminus-lactonized chondroitin sulfateC was dissolved in 200 ml of diethylformamide and 9 mg ofphosphatidylserin stearate palmitate in chloroform was added thereto.The resulting mixture was incubated at 70° C. for 2 hours. Afterremoving chloroform from the reaction mixture by distillation, excessvolume of sodium acetate aqueous solution was added to the residue tomake the reaction product into sodium salt. Sodium acetate-saturatedethanol was added thereto to form a precipitate and the thus-formedprecipitate was collected by filtration. The precipitate was dissolvedin 0.3M ammonium chloride solution and then subjected to purification inaccordance with the procedure of Example 1-(2) to obtain 20.8 mg ofphosphatidylserin stearoylpalmitoyl-linked chondroitin sulfate C (lotNo. 700-2).

Phosphorus content: 0.10%

Chondroitin sulfate C content: 86.15%

Hydrophobic chromatogram: Shown in FIG. 3.

Measuring conditions are the same as described above.

EXAMPLE 3 Preparation of Phospholipid- or Lipid-linked Glycosaminoglycanby Amination of Reducing Terminal Group

(1) Preparation of reducing terminal group-aminated glycosaminoglycan

1) Preparation of reducing terminus-aminated chondroitin sulfate C(CS(S3))

100 mg of reducing terminal group-limitedly oxidized chondroitin sulfateC (lot No. 202-2) was dissolved in 50 ml of 0.05M phosphate buffer (pH7.0), and the solution was mixed with 24 mg of ethylenediaminehydrochloride. After incubating the resulting mixture at 50° C. for 30minutes, 20 mg of sodium cyanoboron hydride was added to the reactionmixture and the incubation was continued at 50° C. for 2 hours tocomplete the reaction. Sodium acetate-saturated ethanol was added to theresulting reaction mixture to precipitate the reaction product which wasthen collected by filtration. The precipitate was dissolved in water andabsorbed to 50 ml of DEAE-ion exchange resin, followed by gradientelution with 0.1M-1M sodium chloride aqueous solution. The reducingterminus-aminated chondroitin sulfate C was eluted with 0.4M sodiumchloride, while free chondroitin sulfate C was eluted with 0.75M sodiumchloride. The 0.4M sodium chloride fraction was desalted by dialysis andthen freeze-dried to obtain 80 mg of reducing terminal group-aminatedchondroitin sulfate C (lot No. 802-2).

2) Preparation of reducing terminus-aminated heparin (Hep)

The above procedure was repeated except that 100 mg of lot No. 205reducing terminal group-limitedly oxidized heparin was used. Thus, 77 mgof reducing terminus-aminated heparin (lot No. 805) was obtained.

(2) Preparation of succinic acid derivative of lipid

1) Preparation of succinic acid ester of glycerol monostearate

10.74 g of glycerol monostearate was dissolved in 200 ml of benzenecontaining 3 ml of pyridine. After adding 6 g of succinic anhydride, theresulting mixture was subjected to reflux for 6 hours. The resultingreaction mixture was concentrated under a reduced pressure, and theprecipitate thus formed was subjected to recrystallization from acetoneto obtain 8.2 g of succinic acid ester of glycerol monostearate.

2) Preparation of active ester from succinic acid ester of glycerolmonostearate with N-hydroxysuccinic acid imide

8 g of the ester obtained in the above procedure 1) was dissolved inbenzene, and the solution was mixed with 2 g of N-hydroxysuccinic acidimide and 10 g of dicyclohexylcarbodiimide. After incubating theresulting mixture at room temperature for 20 hours, the reaction mixturewas concentrated under a reduced pressure to obtain precipitate of thereaction product. The precipitate was recrystallized from abenzene/n-hexane solvent system to obtain 7.4 g of the desired activeester (lot No. GMS-1).

(3) Preparation of glycerol monostearate-linked chondroitin sulfate C##STR98##

In 5 ml of water was dissolved 80 mg of the lot No. 802-2 reducingterminus-aminated chondroitin sulfate C. The resulting solution wasmixed with 6.95 mg of the lot No. GMS-1 active ester dissolved indimethylformamide. After incubating the resulting mixture at roomtemperature for 20 hours, the reaction mixture was mixed with sodiumacetate-saturated ethanol to precipitate the reaction product which wassubsequently collected by filtration. The thus-collected precipitate wasdissolved in 0.3M ammonium chloride aqueous solution and the resultingsolution was subjected to purification in the same manner as in theprocedure of Example 1-(2)-1) to obtain 38 mg of the desired compound(lot No. 902-2).

Stearic acid content: 0.86%

Chondroitin sulfate C content: 98.2%

(4) Preparation of succinic acid derivative of phospholipid

1) Preparation of succinic acid ester of lysolecithin

In 200 ml of chloroform was dissolved 495 mg of lysolecithin of thefollowing formula. ##STR99## To the resulting solution were added 100 mgof succinic anhydride and 79 mg of pyridine. After incubating themixture at room temperature for 20 hours, the reaction mixture wasconcentrated under a reduced pressure to form a precipitate. Thethus-formed precipitate was recrystallized from acetone to obtain asuccinic acid ester of lysolecithin.

2) Preparation of active ester from succinic acid ester of lysolecithinwith N-hydroxysuccinic acid imide

288.5 mg of the ester obtained above was dissolved in dimethylformamide,and the solution was mixed with 57.5 mg of N-hydroxysuccinic acid imideand 103 mg of dicyclohexylcarbodiimide. After incubating thethus-prepared mixture at room temperature for 20 hours, precipitatedmaterials were removed from the resulting reaction mixture to obtain adimethylformamide solution of the desired active ester.

(5) Preparation of lysolecithin-linked glycosaminoglycan

5) Preparation of lysolecithin-linked chondroitin sulfate C ##STR100##

The dimethylformamide solution of the active ester obtained in the aboveprocedure (4)-2) was mixed with an aqueous solution of 1 g of the lotNo. 802-2 reducing terminus-aminated chondroitin sulfate C, and themixture was incubated at room temperature for 20 hours to effect thereaction. Purification of the reaction product was carried out byhydrophobic chromatography in accordance with the procedure of Example1.

Yield: 0.52 g

Phosphorus content: 0.105%

Lysolecithin content: 1.96%

Chondroitin sulfate content: 98.04%

Sulfur content: 5.78%

(6) Preparation of glycerol distearate-linked chondroitin sulfate C

An active ester of a succinic acid ester of glycerol distearate wasprepared according to the above procedure (2)-2) (lot No. GDS-2). Thiswas allowed to react with reducing terminus-aminated chondroitin sulfateC (lot No. 802-2) obtained in the above procedure (1)-1) in accordancewith the above procedure (3), followed by purification. Thus, 27 mg ofthe desired compound was obtained (lot No. 904).

EXAMPLE 4 Preparation of Phospholipid-linked Glycosaminoglycan UsingCondensing Agent

(1) Preparation of L-(α-phosphatidyl)ethanolamine dipalmitoyl-linkedchondroitin sulfate C ##STR101##

400 mg of tri-n-butylamine salt of chondroitin sulfate C (CS(S3)) wasdissolved in 100 ml of dimethylformamide. To the resulting solution wereadded 6.92 mg of PPEADP dissolved in chloroform and 38.4 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Theresulting mixture was incubated at room temperature for 20 hours toeffect the reaction. After concentrating under a reduced pressure,excess volume of sodium acetate aqueous solution was added to theconcentrate to make the reaction product into sodium salt. Ethanol wasadded thereto to precipitate the salt and the thus-formed precipitatewas collected by filtration. The precipitate was dissolved in 0.3Mammonium chloride solution and then subjected to purification inaccordance with the procedure of Example 1-(2)-1). Thus, 63 mg of thedesired compound (lot No. 1002-2) was obtained.

Phosphorus content: 0.099%

PPEADP content: 2.25%

Chondroitin sulfate C content: 96.61%

Hydrophobic chromatogram: Shown in FIG. 4

Measuring conditions are the same as described above.

(2) Preparation of other L-(α-phosphatidyl)ethanolaminedipalmitoyl-linked glycosaminoglycans (GAG-PPEADP)

Phospholipid-linked glycosaminoglycans were prepared from variousglycosaminoglycans and PPEADP in accordance with the above procedure (1)under conditions shown in Table L. Results of the analysis of thethus-obtained products are shown in Table M.

                  TABLE L                                                         ______________________________________                                                            Reaction condition                                        Lot No.                                                                              Product      GAG*.sup.1 /PPEADP/WSC (mg/mg/mg)                         ______________________________________                                        1000   HA1-PPEADP    420/20.76/103                                            1001   CH-PPEADP     420/13.84/68.8                                           1002-1 CS(S1)-PPEADP                                                                               400/20.76/103                                            1002-3 CS(S6)-PPEADP                                                                              400/3.46/17.2                                             1003   CS(2)-PPEADP 400/6.92/34.3                                             1004   DS-PPEADP     40/1.38/6.88                                             1005   Hep-PPEADP   400/13.8/68.8                                             1006   HS-PPEADP    13/0.46/2.3                                               ______________________________________                                         *1: trin-butylamine salt                                                 

                  TABLE M                                                         ______________________________________                                                             Yield    PPEADP GAG                                      Lot No.  Product     (mg)     (%)    (%)                                      ______________________________________                                        1000     HA1-PPEADP  28       6.21   90.05                                    1001     CH-PPEADP   25.8     4.01   88.64                                    1002-1   CS(S1)-PPEADP                                                                             51.9     5.28   92.40                                    1002-3   CS(S6)-PPEADP                                                                             42.2     1.04   97.81                                    1003     CS(W)-PPEADP                                                                              41.9     2.17   96.62                                    1004     DS-PPEADP   29       4.41   89.12                                    1005     Hep-PPEADP  101.3    4.04   90.03                                    1006     HS-PPEADP   1.2      4.00   88.22                                    ______________________________________                                    

EXAMPLE 5 Preparation of Phospholipid-linked Glycosaminoglycan byGlycosaminoglycan-activation Method

(1) Preparation of L-(α-phosphatidyl)ethanolamine dipalmitoyl-linkedchondroitin sulfate C ##STR102##

400 mg of tri-n-butylamine salt of chondroitin sulfate C (CS(S3)) wasdissolved in 300 ml of DMF. To the resulting solution were added 9.9 mgof N-hydroxysuccinimide and 20.6 mg of dicyclohexylcarbodiimide. Theresulting mixture was incubated at room temperature for 20 hours. Excessvolume of sodium acetate aqueous solution was added to the resultingreaction mixture to make the reaction product into sodium salt, followedby the addition of ethanol to collect formed precipitate by filtration.The thus-collected precipitate was immediately dissolved in 30 ml ofwater, and the solution was mixed with 6.92 g of PPEADP dissolved inchloroform. Dimethylformamide was further added thereto to obtain auniform solution. After incubating the thus-obtained solution at roomtemperature for 6 hours, the reaction mixture was concentrated under areduced pressure and then mixed with acetic acid-saturated ethanol toform a precipitate. Thereafter, the thus-formed precipitate wascollected by filtration and dissolved in 0.3M ammonium acetate solutionfollowed by purification in accordance with the procedure of Example1-(2)-1). Thus, 29.7 mg of the desired compound (lot No. 1102-2) wasobtained.

Phosphorus content: 0.100%

PPEADP content: 2.16%

Chondroitin sulfate C content: 95.68%

Hydrophobic chromatogram: Shown in FIG. 5.

Measuring conditions are the same as described above.

(2) Preparation of L-(α-phosphatidyl)ethanolamine dipalmitoyl-linkedchondroitin polysulfate

1 g of tri-n-butylamine salt (sulfur content, 13.0%; molecular weight,10,000) of chondroitin polysulfate (CSP(II)) was dissolved in 50 ml ofdimethylformamide. To the resulting solution were added 1770 mg ofN-hydroxysuccinimide and 318 mg of dicyclohexylcarbodiimide. Theresulting mixture was incubated overnight at 4° C. Thereafter, 10 ml ofwater was added to the reaction mixture and the mixture was furtherallowed to react at room temperature for 15 minutes. After removingformed precipitate, the resulting solution was mixed with 69.2 g ofphosphatidyl ethanolamine dipalmitoyl (PPEADP) dissolved in chloroform,and the mixture was allowed to react at room temperature for 6 hours.The resulting reaction mixture was concentrated under a reduced pressureand then mixed with sodium acetate-saturated ethanol to form aprecipitate. Thereafter, the thus-formed precipitate was collected byfiltration and dissolved in 0.3M ammonium acetate solution, followed bypurification in accordance with the procedure of Example 1-(2)-1). Thus,67 mg of the desired compound (lot No. 1108) was obtained.

Phosphorus content: 0.291%

PPEADP content: 6.5%

Chondroitin polysulfate content: 92.8%

Sulfur content: 12.05%

Hydrophobic chromatogram: Shown in FIG. 6.

Measuring conditions are the same as described above.

EXAMPLE 6 Adhesion of BHK Cells to a Phospholipid- or Lipid-linkedGlycosaminoglycan Layer Laminated on Fibronectin-coated Inside Wall of aCulture Dish

Each well of a 96-well incubation plate was coated with 100 μl of 5μg/ml solution of bovine serum-derived fibronectin. After washing, eachwell was further coated with 100 μl of each of the phospholipid- orlipid-linked glycosaminoglycans obtained in Examples 1 to 5, with theirconcentrations shown in Table N.

Separately, BHK cells (new-born hamster kidney cells) cultured in a dishof 100 mm in diameter were treated with 5 ml of 0.1 mg/ml trypsinsolution at 37° C. for 5 minutes. To the thus-treated cells was added 5ml of 1 μg/ml solution of soy bean trypsin inhibitor in order toinactivate trypsin. Thereafter, the thus-separated cells were collectedby centrifugation, washed twice and then made into a single cellsuspension with a density of 1×10⁵ cells/ml.

A 100 μl portion of the thus-obtained single cell suspension (1×10⁴cells) was poured in each well of the incubation plate which had beendouble-coated with fibronectin and a phospholipid- or lipid-linkedglycosaminoglycan as described above. After incubating at 37° C. for 1hour, cells which did not adhere were washed out, and the remainingadhered cells were fixed with 2% formaldehyde and observed directlyunder a phase-contrast microscope to count the number of adhered cells.

Table N shows the concentration-depending changes in the number ofadhered cells. Each of the data was expressed in terms of a mean valueof three or four measurements. Error (standard deviation) in eachexperiment is also shown.

When free glycosaminoglycans or unlinked lipids were used instead of thephospholipid- or lipid-linked glycosaminoglycans, they showed no celladhesion-inhibiting effect even at high concentrations.

                                      TABLE N                                     __________________________________________________________________________    Lot No.                                                                       Sample                                                                             302-3   600      601     602     602-2   602-3 CS                                                                              604                     (μg/ml)                                                                         CS(S6)-PPEADP                                                                         HA1-PPEADP                                                                             CH-PPEADP                                                                             CS(S1)-PPEADP                                                                         CS(S3)-PPEADP                                                                         (S6)-PPEADP                                                                           DS-PPEADP               __________________________________________________________________________    0.1                                   98.7% ± 0.9%                                                                       98.9% ± 0.9%                 0.2                           87.4% ± 8.8%                                                                       82.4% ± 3.8%                                                                       89.3% ± 0.5%                                                                       85.8% ± 4.6%         0.5                            89.1% ± 15.3%                                                                     49.8% ± 4.6%                                                                       85.0% ± l.1%                                                                       80.2% ± 3.6%         1    91.4% ± 6.8%          50.2% ± 5.2%                                                                       44.7% ± 6.8%                                                                       40.0% ± 3.6%                                                                       -- ± --              2    60.9% ± 4.5%  85.0% ± 2.6%                                                                       33.5% ± 6.9%                                                                       35.4% ± 9.5%                                                                       12.7% ± l.0%                                                                       -- ± --              5    23.0% ± 0.2%                                                                       73.5% ± 1.3%                                                                        81.6% ± 6.0%                                                                       24.4% ± 9.5%                                                                        4.3% ± 1.3%                                                                        1.6% ± 0.7%                                                                       14.9% ± 2.0%         10    1.3% ± 1.2%                                                                       72.5% ± 8.8%                                                                        80.5% ± 6.9%                                                                       12.8% ± 2.4%                                                                        2.8% ± 0.4%                                                                        1.3% ± 0.5%                                                                        6.7% ± 1.2%         20           29.9% ± 2.6%                                                                        63.8% ± 2.7%                                                                        3.2% ± l.1%                                                                        0.7% ± 0.1%                                                                        0.0% ± 0.0%                                                                        2.4% ± 0.9%         50            3.6% ± 0.5%                                                                         65.5% ± 10.0%                0.9% ± 0.6%         100           0.8% ± 0.1%                                                                        44.4% ± 4.2%                                         200           0.3% ± 0.2%                                                                        33.8% ± 3.8%                                         500                                                                           __________________________________________________________________________    Lot No.                                                                       Sample                                                                              605      606       1000     1002-2 CS 1004     1005                     (μg/ml)                                                                          Hep-PPEADP                                                                             HS-PPEADP HA1-PPEADP                                                                             (S3)-PPEADP                                                                             DS-PPEADP                                                                              Hep-PPEAD                __________________________________________________________________________    0.1                                                                           0.2                                                                           0.5                                                                           1                                  88.3% ± 16.6%                           2     86.7% ± 1.3%             57.6% ± 4.8%                                                                         99.3% ± 7.8%                   5     76.1% ± 5.4%                                                                        14.9% ± 0.6%    19.6% ± 4.4%                                                                         89.9% ± 0.4%                   10    60.5% ± 6.9%                                                                        5.7% ± 0.5%      7.5% ± 3.7%                                                                         75.6% ± 4.9%                   20    44.0% ± 5.0%                                                                        2.0% ± 0.3%                                                                          95.6% ± 4.8%    65.0% ± 5.7%                                                                        95.3% ± 0.9%          50    46.0% ± 7.5%                                                                        i.3% ± 0.4%                                                                          80.5% ± 5.4%    56.2% ± 1.1%                                                                         75.0% ± 12.1%        100   17.3% ± 3.4%                                                                        1.0% ± 0.1%                                                                          73.5% ± 0.6%    43.7% ± 4.9%                                                                        55.9% ± 1.1%          200   11.6% ± 2.2%                                                                        0.9% ± 0.0%                                                                          55.0% ± 0.3%             45.1% ± 1.0%          500                                                                           __________________________________________________________________________

EXAMPLE 7 Effect of Phospholipid- or Lipid-linked Glycosaminoglycan forInhibiting Cell Adhesion of Various Cultured Cell Lines by Cell AdhesionSubstances

The phospholipid- or lipid-linked glycosaminoglycans obtained inExamples 1 to 5 were examined for their effects for inhibiting celladhesion of various cell lines by cell adhesion substances, using BHK 21(new-born hamster kidney cell), CEF (arian embryo fibroblast cell),B16F10 (highly metastatic mouse melanoma cell), CHO (Chinese hamsterovarian cell) and baEC (bovine aorta endothelial cell) as the celllines, and fibronectin (FN), laminin (LN), type I collagen (ColI) andvitronectin (VN) as the cell adhesion substances.

A 5 μg/ml portion of each of fibronectin derived from bovine bloodplasma, laminin derived from mouse EHS tumor cell, type I collagenderived from rat thigh and bovine serum-derived vitronectin was coatedon a 96-well incubation plate and each of the phospholipid- orlipid-linked glycosaminoglycans obtained in Examples 1 to 5 was furthercoated in the same manner as in Example 6. Thereafter, a 100 μl portionof a single cell suspension (1×10⁴ cells) of each of BHK 21, CEF,B16F10, CHO and baEC cells was poured in each well to observe changes inthe cell adhesion. As a control, the same procedure was repeated exceptthat the phospholipid- or lipid-linked glycosaminoglycan was not used,and the resulting cell adhesion was expressed as 100%. The results areshown in Table O.

In Table O, relative adhered cell numbers are semi-quantitativelyexpressed as "-" for no or little adhesion (0 to less than 10%), "+" for10 to less than 30%, "++" for 30 to less than 50%, "+++" for 50 to lessthan 70%, "++++" for 70 to less than 90% and "+++++" for 90 to 100%adhesion.

                                      TABLE O                                     __________________________________________________________________________                   Cells/cell adhesion substance (5 μg/ml)                                Amount                                                                            BHK 21  CEF     B16F10  CHO                                    Lot No.    (μg/ml)                                                                        FN  VN  FN  VN  FN  LN  FN  LN                                 __________________________________________________________________________    300(HA1-PPEADP)                                                                          10  +                                                                         100                                                                301(CH-PPEADP)                                                                           10  +++++   ++++                                                              100 +++     +++                                                    302(CS(S1)-PPEADP)                                                                       10  +++ +   ++                                                                100 ++  -   -   -   -   -   -   -                                  302-2(CS(S3)-PPEADP)                                                                     10  -   -   -   -   -   -   -   -                                             100 -   -   -                                                      302-3(CS(S6)-PPEADP)                                                                     10  ++  ++                  ++  +                                             100 +   +                   -   -                                  303(CS(W)-PPEADP)                                                                        10  -   -   -   -   -   -   -   -                                             100 -   -   -   -   -   -   -   -                                  304(DS-PPEADP)                                                                           10  +++                                                                       100                                                                305(Hep-PPEADP)                                                                          10  +++++                                                                     100 +++                                                            600(HA1-PPEADP)                                                                          10  +++                                                                       100                                                                601(CH-PPEADP)                                                                           10  +++             +++                                                       100 +               +                                              602(CS(S1)-PPEADP)                                                                       10  -               -                                                         100 -               -                                              602-2(CS(S3)-PPEADP)                                                                     10  -   -   -   -   -   -   -   -                                             100 -   -   -   -   -   -   -   -                                  702-2(CS(S3-P.S)                                                                         10  -   -   -                                                                 100 -   -   -                                                      602-3(CS(S6)-PPEADP)                                                                     10  -               -                                                         100 -               -                                              604(DS-PPEADP)                                                                           10  -               -                                                         100 -               -                                              605(Hep-PPEADP)                                                                          10  +++             ++                                                        100 +               +                                              606(HS-PPEADP)                                                                           10  +               +                                                         100 -               -                                              902-2(CS(S3)-GMS)                                                                        10  +++++                                                                     100 +++                                                            904(CS(S3)-GDS)                                                                          10  +++                                                                       100 +                                                              __________________________________________________________________________                          Cells/cell adhesion substance (5 μg/ml)                            Amount  baEC                                                    Lot No.       (μg/ml)                                                                            FN          ColI                                        __________________________________________________________________________    600(HA1-PPEADP)                                                                             10      +           ++                                                        100     -           -                                           601(CH-PPEADP)                                                                              10      ++++        ++++                                                      100     ++          ++                                          602(CS(S1)-PPEADP)                                                                          10      -           +                                                         100     -           -                                           602-2(CS(S3)-PPEADP)                                                                        10      -           -                                                         100     -           -                                           602-3(CS(S6)-PPEADP)                                                                        10      -           -                                                         100     -           -                                           604(DS-PPEADP)                                                                              10      -           ++                                                        100     -           -                                           605(Hep-PPEADP)                                                                             10      ++          +++                                                       100     +           ++                                          606(HS-PPEADP)                                                                              10      ++          ++                                                        100     -           -                                           HA1           100     +++++       +++++                                       CH            100     +++++       +++++                                       CS(S1)        100     +++++       +++++                                       CS(S3)        100     +++++       +++++                                       CS(S6)        100     +++++       +++++                                       DS            100     +++++       +++++                                       Hep           100     +++++       +++++                                       HS            100     +++++       +++++                                       PPEADP        100     +++++       +++++                                       __________________________________________________________________________                    Cells/cell adhesion substance (5 μg/ml)                                Amount                                                                            BHK 21  CEF     B16F10  CHO                                   Lot No.     (μg/ml)                                                                        FN  VN  FN  VN  FN  LN  FN  LN                                __________________________________________________________________________    1000(HA1-PPEADP)                                                                          10  +++++                                                                     100 ++++                                                          1001(CH-PPEADP)                                                                           10  +++++   ++++                                                              100 +++     +++                                                   1002(CS(S1)-PPEADP)                                                                       10  +++ +   ++                                                                100 ++  -   -                                                     1002-2(CS(S3)-PPEADP)                                                                     10  -   -   -   -   -   -   -   -                                             100 -   -   -   -   -   -   -   -                                 1002-3(CS(S6)-PPEADP)                                                                     10  ++  ++                                                                    100 +   +                                                         1003(CS(W)-PPEADP)                                                                        10  -   -   -                                                                 100 -   -   -                                                     1004(DS-PPEADP)                                                                           10  ++++                                                                      100 ++                                                            1005(Hep-PPEADP)                                                                          10  +++++                                                                     100 +++                                                           1006(HS-PPEADP)                                                                           10  ++                                                                        100                                                               1108(CPS(II)-PPEADP)                                                                      10          +++                                                               100         +                                                     HA1         100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             HA5         100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             HA15        100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             CH          100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             CS(S1)      100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             CS(S3)      100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             CS(S6)      100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             CS(W)       100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             DS          100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             Hep         100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             HS          100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             KS          100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             CPS(II)     100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             PPEADP      100 +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                                                                             +++++                             __________________________________________________________________________

EXAMPLE 8 Effect of Phospholipid-linked Chondroitin Sulfate C forInhibiting Adhesion of Highly Metastatic Cancer Cells to ExtracellularMatrix of Blood Vessel Endothelial Culture Cells

Mouse blood vessel endothelial cells were cultured in a 24-wellincubation plate which had been coated with type I collagen so that thecells grew confluent. Single layer of the cells was treated with 0.5%Triton X-100 at room temperature for 30 minutes, and thethus-disintegrated fragments of the cell layer were washed withDulbecco's PBS(+) buffer to obtain extracellular matrix of endothelialcells.

Separately, mouse-derived highly metastatic cancer cells (B16F10)cultured in a dish of 100 mm in diameter were treated with 5 ml of atrypsin solution (0.1 mg/ml PBS(-)) at 37° C. for 5 minutes. To thethus-treated cells was added 5 ml of 1 mg/ml solution of soy beantrypsin inhibitor in order to inactivate trypsin. Thereafter, thethus-separated cells were collected by centrifugation, washed twice witha phosphate buffer (PBS (-)) and then made into a single cell suspension(Hanks' BSS--20 mM HEPES, pH 7.4) with a density of 2×10⁵ cells/ml.

The lot No. 602-2 phospholipid-linked chondroitin sulfate C(CS(S3)-PPEADP) and 500 μl of the thus-prepared single cell suspensionof B16F10 (1×10⁵ cells) were transferred into each well of theaforementioned extracellular matrix-containing 24-well incubation plateand incubated statically at 37° C. for 1 hour in an incubator chargedwith 5% carbon dioxide.

The supernatant fluid in each well was taken out gently, the residue waswashed once gently with Hanks' buffer and both liquid portions werecombined. Thereafter, the number of cells in the combined sample wascounted using a cell counter (Coulter Electronics) to count cells whichdid not adhered to the extracellular matrix. A buffer solutioncontaining no lot No. 602-2 compound (no addition) and a buffercontaining free chondroitin sulfate C were used as controls.

Adhesion ratio of cells was calculated by subtracting the number ofcounted unadhered cells from the initially added total cells anddividing the remainder by the number of total cells. The results areshown in Table P.

                  TABLE P                                                         ______________________________________                                        Sample           Amount added                                                                             Adhesion ratio                                    ______________________________________                                        No addition      --         82.8%                                             Free chondroitin sulfate C                                                                     50 μg   82.6%                                             602-2 (CS(S3)-PPEADP)                                                                          50 μg   50.7%                                             ______________________________________                                    

As is evident from the above results, the phospholipid-linkedglycosaminoglycan of the present invention can inhibit adhesion ofhighly metastatic cancer cells to extracellular matrices of blood vesselendothelial cells, while free chondroitin sulfate C cannot exhibit suchan effect.

EXAMPLE 9 Effect of Phospholipid-linked Chondroitin Sulfate C forInhibiting Metastasis of Highly Metastatic Cancer Cells

Mouse-derived highly metastatic cancer cells (B16F10) cultured in a dishof 100 mm in diameter were treated with 5 ml of an EDTA solution(0.02%/PBS (-)) at 37° C. for 5 minutes, followed by cell separation bypipetting. The cells were collected by centrifugation, washed twice witha phosphate buffer (PBS (-)) and then made into a single cell suspensionwith a density of 1×10⁻⁶ cells/ml. A 0.1 ml portion of this cellsuspension (10⁵ cells) was mixed with 0.1 ml of a PBS solution of thelot No. 602-2 (CS(S3)-PPEADP) phospholipid-linked glycosaminoglycan (0.1mg, 1 mg or 5 mg/0.1 ml) and the resulting mixture was administered toC57BL/6 mice through tail vein. Thoracotomy was carried out 2 weeksafter the administration to count the number of melanoma coloniesmetastasized to the surface of the lungs. A buffer solution containingno lot No. 602-2 (CS(S3)-PPEADP) compound (no addition) and a buffercontaining free chondroitin sulfate C were used as controls.

Table Q shows the results of counting colonies metastasized to thesurface of the lungs per mouse.

                  TABLE Q                                                         ______________________________________                                                          Dose      Colonies                                          Sample            (mg/mouse)                                                                              (numbers/mouse)                                   ______________________________________                                        No addition       --        49.9 ± 32.3                                    Free chondroitin sulfate C                                                                      5 mg      24.0 ± 8.0                                     602-2 (CS(S3)-PPEADP)                                                                           0.1 mg    47.2 ± 28.1                                                      1 ml      22.0 ± 14.8                                                      5 mg      4.0 ± 3.5                                      ______________________________________                                    

Metastasis of cancer cells was inhibited as the dose of thephospholipid-linked chondroitin sulfate C increased. As is evident fromthese results, the phospholipid-linked glycosaminoglycan of the presentinvention can inhibit metastasis of highly metastatic cancer cells. Freechondroitin sulfate C also inhibited the metastasis, but its effect wasinferior to that of the phospholipid-linked chondroitin sulfate C(p<0.001).

INDUSTRIAL APPLICABILITY

The phospholipid- or lipid-linked glycosaminoglycans or their salts ofthe present invention which has cell adhesion-inhibitory activity andhas no toxicity are useful as metastasis inhibitors.

What is claimed is:
 1. A metastasis inhibitor composition comprising:(A)a phospholipid-linked glycosaminoglycan or a pharmaceutically acceptablesalt thereof, in which a primary amino group of a phospholipid is linkedto a carbonyl group of a reducing terminal moiety of a glycosaminoglycanthrough an amido bond, said phospholipid-linked glycosaminoglycan beingrepresented by one of the following formulae: ##STR103## wherein P¹ is aphospholipid represented by the formula (IX): ##STR104## wherein each ofR⁴ and R⁵ is hydrogen, --CH═CHR⁶ or --COR⁷, wherein each of R⁶ and R⁷ isa C₆₋₂₄ alkyl group, and Y is --CH₂ CH₂ NH-- or ##STR105## and GAG is aglycosaminoglycan residue of hyaluronic acid or chondroitin sulfate,wherein the reducing terminal group has been removed, and (B) apharmaceutically acceptable carrier or diluent.
 2. A metastasisinhibitor composition comprising:(A) a phospholipid- or lipid-linkedglycosaminoglycan or a pharmaceutically acceptable salt thereof, inwhich an oxygen atom of a hydroxyl group of a phospholipid- or a lipidis linked to a terminal carbonyl group of a group represented by--NH--(CH₂)_(m) --NHCO--(CH₂)_(l) --CO-- though an ester bond, and analdehyde residue of a reducing terminal moiety of a glycosaminoglycan islinked to a terminal --NH group of a group represented by--NH--(CH₂)_(m) --NHCO--(CH₂)_(l) --CO-- through a CH₂ NH bond, saidphospholipid- or lipid-linked glycosaminoglycan being represented by oneof the following formulae: ##STR106## wherein R represents--NH--(CH₂)_(m) --NHCO--(CH₂)_(l) --CO--P², wherein P² is a phospholipidor a lipid represented by one of the following formulae: ##STR107##wherein R⁸ is hydrogen, R⁹ is an alkyl group, R¹⁰ is --CH═CHR⁶ or--COR⁷, wherein each of R⁶ and R⁷ is a C₆₋₂₄ alkyl group, m is aninteger of 1 to 8 and l is an integer of 1 to 10, and X is --CH₂ CH₂ N⁺(CH₃)₃ or an inositol residue, and GAG is a glycosaminoglycan residue ofhyaluronic acid or chondroitin sulfate, and (B) a pharmaceuticallyacceptable carrier or diluent.
 3. A metastasis inhibitor compositioncomprising:(A) a phospholipid-linked or lipid-linked glycosaminoglycanor a pharmaceutically acceptable salt thereof, in which an oxygen atomof a hydroxyl group or a phospholipid or a lipid is linked to a terminalcarbonyl group of a group represented by --NH--(CH₂)_(m)--NHCO--(CH₂)_(l) --CO-- through an ester bond, and a carbonyl group ofa reducing terminal moiety of a glycosaminoglycan is linked to aterminal --NH group of a group represented by --NH--(CH₂)_(m)--NHCO--(CH₂)_(l) --CO-- through an amido bond, said phospholipid- orlipid-linked glycosaminoglycan being represented by one of the followingformulae: ##STR108## wherein R represents --NH--(CH₂)_(m)--NHCO--(CH₂)_(l--CO--P) ², wherein P² is a phospholipid or a lipidrepresented by one of the following formulae: ##STR109## wherein R⁸ ishydrogen, R⁹ is an alkyl group, R¹⁰ is --CH═CHR⁶ or --COR⁷, wherein eachor R⁶ and R⁷ is a C₆₋₂₄ alkyl group, m is an integer of 1 to 2, and l isan integer of 1 to 10, and X is --CH₂ CH₂ N⁺ (CH₃) or an inositolresidue and GAG is a glycosaminoglycan residue of hyaluronic acid orchondroitin sulfate, and (B) a pharmaceutically acceptable carrier ordiluent.
 4. A metastasis inhibitor composition comprising:(A) aphospholipid-linked glycosaminoglycan or a pharmaceutically acceptablesalt thereof, in which a primary amino group of a phospholipid is linkedto a carboxyl group of a uronic acid moiety of a glycosaminoglycan, saidphospholipid-linked glycosaminoglycan being represented by the one offollowing formulae: ##STR110## wherein P¹ is a phospholipid representedby the formula (IX): ##STR111## wherein each of R⁴ and R⁵ is hydrogen,--CH═CHR⁶ or --COR⁷, wherein each or R⁶ and R⁷ is a C₆₋₂₄ alkyl group,and Y is --CH₂ CH₂ NH-- or ##STR112## and GAG is a glycosaminoglycanresidue of hyaluronic acid or chondroitin sulfate, wherein the reducingterminal group has been removed, and (B) a pharmaceutically acceptablecarrier or diluent.
 5. The metastasis inhibitor composition according toany of one of claims 1, 2, 3, or 4, wherein said glycosaminoglycan isselected from the group consisting of chondroitin sulfate A, B, C, D, Eor K, and chondroitin polysulfate, and said phospholipid isphosphatidylethanolamine or phosphatidylserine.
 6. A method ofinhibiting metastasis of metastatic cancer cells, which comprisesadministering to a mammal suffering from cancer a pharmaceuticallyeffective amount of a phospholipid-linked glycosaminoglycan compositionaccording to claim
 1. 7. A method of inhibiting metastasis of metastaticcancer cells, which comprises administering to a mammal suffering fromcancer a pharmaceutically effective amount of a phospholipid-linkedglycosaminoglycan composition according to claim
 5. 8. A method ofinhibiting metastasis of metastatic cancer cells, which comprisesadministering to a mammal suffering from cancer a pharmaceuticallyeffective amount of a phospholipid-linked glycosaminoglycan compositionaccording to claim
 2. 9. A method of inhibiting metastasis of metastaticcancer cells, which comprises administering to a mammal suffering fromcancer a pharmaceutically effective amount of a phospholipid-linked orlipid-linked glycosaminoglycan composition according to claim
 3. 10. Amethod of inhibiting metastasis of metastatic cancer cells, whichcomprises administering to a mammal suffering from cancer apharmaceutically effective amount of a phospholipid-linkedglycosaminoglycan composition according to claim
 4. 11. A method ofinhibiting metastasis of metastatic cancer cells, which comprisesadministering to a mammal suffering from cancer a pharmaceuticallyeffective amount of a lipid-linked glycosaminoglycan, wherein the lipidis covalently bonded to said glycosaminoglycan directly or through alinker moiety represented by --NH--(CH₂)_(m) --NHCO--(CH₂)_(l) --CO--,wherein said glycosaminoglycan is a member selected from the groupconsisting of hyaluronic acid and chondroitin sulfate, and wherein saidlipid is represented by one of the following formulae: ##STR113##wherein each of R⁴ and R⁵ is hydrogen, --CH═CHR⁶ or --COR⁷, wherein eachor R⁶ and R⁷ is a C₆₋₂₄ alkyl group, and Y is --CH₂ CH₂ NH-- or##STR114## wherein R⁸ is hydrogen, R⁹ is an alkyl group, R¹⁰ is--CH═CHR⁶ or --COR⁷, wherein each or R⁶ and R⁷ is a C₆₋₂₄ alkyl group, mis an integer of 1 to 2, and l is an integer of 1 to 10, and X is --CH₂CH₂ N⁺ (CH₃) or an inositol residue.
 12. The method according to claim11, wherein said glycosaminoglycan is selected from the group consistingof chondroitin sulfate A, B, C, D, E or K, and chondroitin polysulfate,and said phospholipid is phosphatidylethanolamine or phosphatidylserine.13. A method for inhibiting melanoma cell adhesion in vitro, comprisingcontacting melanoma cells in vitro with an effective amount of alipid-linked glycosaminoglycan, wherein the lipid is covalently bondedto said glycosaminoglycan directly or through a linker moietyrepresented by --NH--(CH₂)_(m) --NHCO--(CH₂)_(l) --CO--, wherein saidglycosaminoglycan is a member selected from the group consisting ofhyaluronic acid, chondroitin sulfate, chondroitin polysulfate, dermatansulfate, heparin, heparan sulfate, keratan sulfate, and keratanpolysulfate, and wherein said lipid is represented by one of thefollowing formulae: ##STR115## wherein each of R⁴ and R⁵ is hydrogen,--CH═CHR⁶ or --COR⁷, wherein each or R⁶ and R⁷ is a C₆₋₂₄ alkyl group,and Y is --CH₂ CH₂ NH-- or ##STR116## wherein R⁸ is hydrogen, R⁹ is analkyl group, R¹⁰ is --CH═CHR⁶ or --COR⁷, wherein each or R⁶ and R⁷ is aC₆₋₂₄ alkyl group, m is an integer of 1 to 2, and l is an integer of 1to 10, and X is --CH₂ CH₂ N+(CH₃) or an inositol residue.